Agilent 2100 Bioanalyzer 2100 Expert User’s Guide Edition November 03 s1
Notices Warranty © Agilent Technologies, Inc. 2000-2003 No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or translation into a foreign language) without prior agreement and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws.
Contents About this Manual .................................................................................................................. 6 In this Manual............................................................................................................................. 7 Related Documents ................................................................................................................... 9 What’s New in 2100 Expert.................................................................
Result Flagging ....................................................................................................................... 145 Running and Evaluating Flow Cytometric Assays........................................................... 156 Principles of Flow Cytometric Measurements .................................................................. 157 Overview of Flow Cytometric Assays .................................................................................
How to Use the Advanced Settings .................................................................................... 299 Running Instrument Diagnostics ...................................................................................... 303 How to Run Instrument Diagnostics Tests........................................................................ 308 Performing Qualifications..................................................................................................
About this Manual Welcome to the User’s Guide for the Agilent 2100 expert software. This manual provides beginners and advanced users with information needed to successfully run electrophoretic and flow cytometric assays with the bioanalyzer. The 2100 expert software allows the control of the bioanalyzer (including diagnostic functions) and, in combination with a LabChip kit, the acquisition, interpretation and result presentation of data generated during the analysis of DNA, RNA, proteins, and cells.
In this Manual This manual provides bioanalyzer users with the following information: • “About this Manual” on page 6 gives an overview of the subjects in this manual, and lists major innovations and improvements of the 2100 expert software. It also lists supplemental literature and shows you how to make efficient use of this manual. • “Quick Start” on page 17 is meant for experienced users. It briefly summarizes the necessary steps to prepare and run an assay.
• “Configuring 2100 Expert” on page 293 is your guideline for configuring the 2100 expert software. • “Running Instrument Diagnostics” on page 303 shows how to use the diagnostics tests to check the bioanalyzer hardware for proper functioning. • “Performing Qualifications” on page 312 describes how you can validate your bioanalyzer system. • “2100 Expert Software Reference” on page 320 describes all elements of the 2100 expert user interface, such as screen regions, menus, tool bars, and dialog boxes.
Related Documents A collection of supplemental literature is given in the following. Bioanalyzer Manuals Publication Number Title G2938-90006 Agilent 2100 Bioanalyzer Installation and Safety Manual G2946-90001 Agilent 2100 Bioanalyzer Maintenance and Troubleshooting Guide CD-ROM Publication Number Title G2946-60002 Agilent 2100 Bioanalyzer – How to Use Multimedia CD-ROM Reagent Kit Guides The Reagent Kit Guides give you information on how to prepare samples.
Publication Number Title G2938-90030 Reagent Kit Guide RNA 6000 Nano Assay G2938-90040 Reagent Kit Guide RNA 6000 Pico Assay G2938-90050 Reagent Kit Guide Protein 200 Plus Assay G2938-90060 Reagent Kit Guide Protein 50 Assay G2938-90070 Reagent Kit Guide Cell Fluorescence Assays G2938-90080 Reagent Kit Guide Cell Fluorescence Checkout Kit Application Notes and Technical Notes Application Notes and Technical Notes are available from the Agilent 2100 Bioanalyzer Help Desk or from the lab-on-a-ch
What’s New in 2100 Expert Products 2100 expert is the successor to the Bio Sizing and Cell Fluorescence software. • 2100 expert provides a single software platform with a common user interface for running, analyzing, evaluating, presenting, and comparing DNA, RNA, protein and cell parameters. • 2100 expert is installed in one go. After installation, the functionality for electrophoretic and flow cytometric assays can be activated separately with license keys.
• 2100 expert has improved instrument control. Two bioanalyzers can be controlled at one time. It is possible to run measurements as well as diagnostics tests on two bioanalyzers at the same time. • 2100 expert has improved printing and reporting functions. • 2100 expert has extended instrument diagnostics functionality.
How to Use this Manual This manual uses convenient online navigation features and follows certain typographic conventions. Online Navigation Use the interactive bookmarks in this frame to move to your desired topic. Use Acrobat Reader’s navigation bar to move around within a topic. Click here to go to the table of contents. Click here to go to the index. Here is the current page number. ▲ Displays the previous page. ▼ Displays the next page.
After you have chosen a topic with the bookmarks, use the buttons in Acrobat Reader’s tool bar to move around within the topic. Displays the next page. Returns to the previous view. Click several times to undo more view changes. Displays the previous page. Displays the first page. Displays the last page. Contents ▲ 14 ▼ Returns to the next view. Click several times to redo more view changes.
Layout Conventions The following typographic conventions are used in this manual: Highlight Meaning Italic On-screen element Example: ... the OK button. Emphasis Example: Right-click the ... Term Example: Dot plots show events as dots. Reference to another document Example: Refer to the Agilent 2100 Bioanalyzer Troubleshooting and Maintenance Guide. Blue Cross-reference or hyperlink Examples: “What’s New in 2100 Expert” on page 11 http://www.agilent.com/chem/labonachip Courier Code Example: ..
Safety Notices, Notes and Tips Safety notices, notes and tips in this document have the following meaning: WA R N I N G A warning notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in personal injury or death. Do not proceed beyond a warning notice until the indicated conditions are fully understood and met. C A U T IO N A caution notice denotes a hazard.
Quick Start The following step-by-step instructions summarize the basic steps needed to perform a measurement with the Agilent 2100 bioanalyzer. Preparing the Agilent 2100 Bioanalyzer 1 Ensure that the proper cartridge is installed in the bioanalyzer. You can identify the installed cartridge by the number engraved in its front. Engraved number Note that there are also electrode cartridges without an engraved number.
Switching on the Agilent 2100 Bioanalyzer 1 Make sure the bioanalyzer is connected to line power and connected to the PC. 2 Turn on the line switch at the rear of the instrument. The status LED at the front of the bioanalyzer should light up. Lid Status LED The status LED shows you the current status of the instrument. Signal Meaning Green light Instrument is switched on and ready for measurement. Green blinking Measurement is running.
Running a Measurement 1 To start the 2100 expert software on the connected PC, go to your desktop and double-click the following icon: After startup of the software, you enter the Instrument context: Contents ▲ 19 ▼ Index
In the upper left of the Instrument tab, an icon shows the status of the bioanalyzer: Icons Meaning Bioanalyzer detected, lid is open. Bioanalyzer detected. Lid is closed, but no chip is inserted. No bioanalyzer has been detected. Check the COM Port setting (see figure under step 3), the RS 232 connection cable, the power cable, and the power switch. For details on how to set up the bioanalyzer and connect it to a PC, see Agilent 2100 Bioanalyzer Installation and Safety Guide.
3 Select an assay for the chip run. On the Instrument tab, click the Assay... button. – OR – Click the Assays menu. Both will open the Assays menu, allowing you to select an assay from the submenus. – OR – You can also select File > Open File to Run.... This opens a dialog box allowing you to load either an assay (.xsy) or a chip data file (.xad).
4 Prepare the samples and the chip. For detailed information on sample and chip preparation refer to the – Reagent Kit Guides that are available for each reagent kit. – Application Notes that are available for each assay. NOTE When preparing chip and samples, pay attention to the essential measurement practices described in “Essential Measurement Practices (Electrophoretic Assays)” on page 67 and “Essential Measurement Practices (Flow Cytometric Assays)” on page 178.
c Place the chip into the receptacle. The figure shows this for an electrophoresis chip. Chip The chip fits only one way. Do not use force. C A U T IO N Do not force the lid closed. This may damage the cartridge. d Carefully close the lid. Electrophoretic assays: the electrodes in the cartridge fit into the wells of the chip. Flow cytometric assays: the adapter with the gasket in the cartridge fits onto the priming well of the chip.
The bioanalyzer icon changes to a chip icon (a DNA chip icon, for example): If the chip is not detected, open and close the lid again. NOTE If the AutoRun option is active (see “Options – Advanced” on page 508), the chip run starts automatically once a chip has been inserted and the lid has been closed. 5 On the Instrument tab, click the Start button.
The chip run starts. The Raw Signals sub-tab shows an electropherogram of the currently measured sample. The name of the sample is displayed above the graph. The graph is a “live” plot of the migration time against fluorescence units (raw data, including background fluorescence, for example).
The number of the sample that is currently being measured is indicated on the information bar: The status bar shows the name of the currently measured sample, a progress bar showing the measurement progress for the current sample (not for the whole chip run), and the COM port number used for data acquisition: During the chip run, you can do the following: • View the chip data file in the Data and Assay context by clicking on the name of the Data File: • Switch to any other context.
Finishing the Measurement When the measurement is finished, the End of run dialog box appears, showing you the status of the assay and the file name where the chip run data has been stored. 1 To immediately view the results, select the Select file in Data Context check box, and click OK.
This takes you to the Data and Assay context, and the data file that has just been generated by your chip run is selected. The Chip Summary tab shows information on your chip data file, and lets you enter comments regarding chip, samples, and study.
2 In the tree view panel, click any sample name, or the ladder. This selects the Electropherogram tab, which displays a data plot of migration time versus fluorescence intensity. Peaks have automatically been detected, and their characteristics such as size, concentration, and molarity have been calculated and are shown in the Peak Table at the bottom of the window.
What You Can do When the Measurement is Finished When the measurement is finished, you can: • Document your chip run by entering sample names, chip comments, and study information, for example.
Looking at 2100 Expert Before you begin with running assays on the Agilent 2100 bioanalyzer you should familiarize yourself with the 2100 expert software: • “Starting 2100 Expert” on page 32 • “2100 Expert Work Area” on page 33 • “Running a Demo Assay” on page 41 • “Closing 2100 Expert” on page 45 Contents ▲ 31 ▼ Index
Starting 2100 Expert To start 2100 expert: 1 Go to your desktop and double-click the following icon: – OR – From the Windows Start menu, select Programs > Agilent 2100 Bioanalyzer > 2100 expert. The 2100 expert application window appears. “2100 Expert Work Area” on page 33 gives an overview of the application window.
2100 Expert Work Area The 2100 expert work area has standard elements such as pull-down menus and toolbars, and the main working area, which contains several tabs, some of which have sub-tabs. The 2100 expert work area has the following regions (Data and Assay context): Title Bar Menu Bar Toolbars Info Bar Context Bar Tree View Tabs Sub-tabs Lower Panel Status Bar Setpoint Explorer See “2100 Expert Application Window Elements” on page 321 for details on the regions.
The 2100 expert software can be operated in four modes, called “contexts”: • Instrument Context • Data and Assay Context • Validation Context • Comparison Context NOTE The contexts are independent from each other regarding their data. This means, for example, that you can review data and run measurements at the same time.
NOTE Menus, toolbars, the tree view, and the main working area (tabs) significantly change when you switch between the contexts. An introduction to the four contexts is given in the following.
Instrument Context On startup, 2100 expert enters the Instrument context, where you can run DNA, RNA, protein or cell assays by selecting an assay file and starting the chip run—provided that the bioanalyzer is properly connected, a chip is inserted, and the bioanalyzer lid is closed.
NOTE If two bioanalyzers are connected to your PC, you can run both in parallel. During the chip run(s), you can view the status of the bioanalyzer(s): instrument information and real time acquisition data. In the Instrument context, it is also possible to run hardware diagnostic tests on all connected bioanalyzers. Refer to “Running Instrument Diagnostics” on page 303 for details.
Data and Assay Context In the Data and Assay context, you can • view, analyze, and evaluate the results of your chip runs that are presented as electropherograms, gel-like images, histograms, dot plots, and result tables. • export and print the results of your chip runs. • modify existing assays and create your own assays by modifying properties such as data analysis setpoints.
Validation Context The Validation context is used to run and document qualification tests. For both the bioanalyzer hardware and software tests can be run for: • Installation qualification (IQ) • Operational qualification (OQ) Validation results are automatically saved in .xvd files. You can re-open .xvd files to review validation results. For details, refer to “Performing Qualifications” on page 312.
Comparison Context In the Comparison context, you can open multiple electrophoretic chip data files and compare samples of the same assay class (DNA 1000, for example), even from different chip runs. It is possible to overlay electropherograms recorded by the bioanalyzer, and compare the measurement results. Comparison results can be saved in .xac files. You can re-open .xac files to review the comparison results, and to add further samples for comparison, for example.
Running a Demo Assay 2100 expert provides demo assays that you can use for a first impression of chip runs and data evaluation, even if the bioanalyzer is offline. To run a demo assay: 1 Start the software as described in “Starting 2100 Expert” on page 32, or, if the software is already running, switch to the Instrument context.s 2 On the Instrument tab, select “Demo” as the COM Port. 3 Click the Assay... button.
This opens a menu, allowing you to select an assay from submenus. 4 Select a demo assay, for example Demo > electrophoresis > Demo Protein 200 Plus. The assay is loaded and a violet chip icon appears indicating the assay type “Protein”. 5 Click the Start button.
The demo chip run starts and you can watch its progress on the Raw Signals tab, where you can see a simulation of data acquisition. For details on how a chip run proceeds, refer to “Running an Electrophoretic Assay” on page 72, or “Running a Flow Cytometric Assay” on page 182. NOTE Like a real chip run, a demo chip run also creates a chip data (.xad) file.
At the end of the simulation, the End of run dialog box appears. 6 Select the Select file in Data Context check box and click OK. This takes you to the Data and Assay context where you can view the results and practice data analysis and evaluation as described in “Analyzing and Evaluating the Results of an Electrophoretic Assay” on page 91 and “Analyzing and Evaluating the Results of a Flow Cytometric Assay” on page 198.
Closing 2100 Expert To close 2100 expert: 1 From the File menu, select Exit. 2100 expert quits. However, if a chip run is in progress, the following message appears: Click OK and wait until the chip run is complete.
Also the following dialog box may appear: NOTE This dialog box may also appear if you try to switch between contexts but there is unsaved data. – Click Yes to save the changes to the selected files and continue quitting 2100 expert. If you want to save changes only to particular files, select these files in the list by single-clicking them. By default, all files with unsaved changes are selected. – If you click No, 2100 expert quits without saving any changes.
Switching Between Electrophoretic and Flow Cytometric Assays The Agilent 2100 bioanalyzer supports electrophoretic assays (DNA, RNA, and protein) and flow cytometric assays (apoptosis, for example).
• The electrode cartridge contains 16 electrodes that fit into the wells of DNA, RNA, and Protein chips. Each electrode in the cartridge has an individual power supply. All electrophoretic assays (DNA, RNA, and Protein) require an electrode cartridge. The electrode cartridge can be identified by the engraved number “1” on the front. • The pressure cartridge contains a tubing and filter assembly that connect to the vacuum pump.
How to Prepare the Bioanalyzer for Electrophoretic Assays Remove the pressure cartridge: 1 Turn off line power to the Agilent 2100 bioanalyzer. The line switch is located at the rear. 2 Open the lid. 3 Pull down the metal locking lever as shown in the figure below. Metal lever in open position The cartridge is pushed out. 4 Gently pull the cartridge out of the lid. NOTE Store the pressure cartridge in the provided box.
Insert the electrode cartridge: C A U T IO N Do not touch the electrodes while the cartridge is in the Agilent 2100 bioanalyzer. The electrodes and the high voltage power supplies can be damaged. 1 Slide the electrode cartridge in the lid as shown below. Push here to ensure tight connection Metal lever 2 Push the metal front of the cartridge to ensure a tight connection. 3 Push the metal locking lever in the flat (closed) position. C A U T IO N Do not force the lid closed. This may damage the cartridge.
4 Remove any chip. C A U T IO N Do not force the chip selector handle when a chip is inserted in the bioanalyzer. 5 Adjust the chip selector to position “1” as shown in the following figure. To avoid using incompatible chips and cartridges, a chip selector is installed in the bioanalyzer. This ensures that the chip matches to the installed cartridge. Move chip selector in position “1” This will allow you to insert DNA, RNA, and Protein chips in the bioanalyzer.
How to Prepare the Bioanalyzer for Flow Cytometric Assays Remove the electrode cartridge: 1 Turn off line power to the Agilent 2100 bioanalyzer. The line switch is located at the rear. 2 Open the lid. C A U T IO N Do not touch the electrodes while the cartridge is in the Agilent 2100 bioanalyzer. The electrodes and the high voltage power supplies can be damaged. 3 Pull down the metal locking lever in the open position as shown in the figure below. Metal lever in open position The cartridge is pushed out.
C A U T IO N Store the electrode cartridge in the provided box. If the pins of the electrode cartridge are bent or misaligned, poor quality results or pre-terminated chip runs will result. Insert the pressure cartridge: 1 Slide the pressure cartridge in the lid as shown below. Push here to ensure tight connection Metal lever 2 Push the metal front of the cartridge to ensure a tight connection. 3 Push the metal locking lever in the flat (closed) position. C A U T IO N Do not force the lid closed.
4 Remove any chip. C A U T IO N Do not force the chip selector handle when a chip is inserted in the bioanalyzer. 5 Adjust the chip selector to position “2” as shown in the following figure. To avoid using incompatible chips and cartridges, a chip selector is installed in the bioanalyzer. This ensures that the chip matches to the installed cartridge. Move chip selector in position “2” This will allow you to insert cell chips in the bioanalyzer.
Running and Evaluating Electrophoretic Assays For running and evaluating electrophoretic assays you need to know the following: • “Principles of Nucleic Acid and Protein Analysis on a Chip” on page 56 • “Preparing and Running an Electrophoretic Assay” on page 60 • “Analyzing and Evaluating the Results of an Electrophoretic Assay” on page 91 • “Result Flagging” on page 145 Contents ▲ 55 ▼ Index
Principles of Nucleic Acid and Protein Analysis on a Chip The electrophoretic assays are based on traditional gel electrophoresis principles that have been transferred to a chip format. The chip format dramatically reduces separation time and sample consumption. The system provides automated sizing and quantitation information in a digital format. On-chip gel electrophoresis is performed for the analysis of DNA, RNA and proteins.
RNA are electrophoretically driven by a voltage gradient—similar to slab gel electrophoresis. Because of a constant mass-to-charge ratio and the presence of a sieving polymer matrix, the molecules are separated by size. Smaller fragments are migrating faster than larger ones. Dye molecules intercalate into DNA or RNA strands or Protein-SDS micells. These complexes are detected by laser-induced fluorescence. Data is translated into gel-like images (bands) and electropherograms (peaks).
The 2100 expert software plots fluorescence intensity versus migration time and produces an electropherogram for each sample: Contents ▲ 58 ▼ Index
The data can also be displayed as a densitometry plot, creating a gel-like image: Contents ▲ 59 ▼ Index
Preparing and Running an Electrophoretic Assay An electrophoretic chip run requires the following steps: 1. Set up and switch on the Agilent 2100 bioanalyzer. Refer to “Switching Between Electrophoretic and Flow Cytometric Assays” on page 47. 2. Start the 2100 expert software. Details are given in “Starting 2100 Expert” on page 32. 3. Select an electrophoretic assay. See “Selecting an Electrophoretic Assay for a Chip Run” on page 62. 4. Prepare chip and samples.
• Analyze and evaluate the results: – “Analyzing and Evaluating the Results of an Electrophoretic Assay” on page 91 – “Result Flagging” on page 145 Contents ▲ 61 ▼ Index
Selecting an Electrophoretic Assay for a Chip Run To select an assay: 1 Switch to the Instrument context. 2 In the Tree View Panel, select the bioanalyzer you want to use. In the upper left of the Instrument tab, an icon shows the status of the bioanalyzer.
3 If you do not see one of these icons, check that the bioanalyzer is switched on and properly connected: – Check the COM port setting. – Make sure the bioanalyzer is physically connected to the PC (over the serial interface). – Check the power connection. – Check the power switch. If you need additional help, please refer to the Agilent 2100 Bioanalyzer Maintenance and Troubleshooting Guide. 4 Select an assay for the chip run. On the Instrument tab, click the Assay... button. – OR – Click the Assays menu.
Both will open the Assays menu, allowing you to select an assay from the submenus. – OR – Select File > Open File to Run.... This opens a dialog box, allowing you to load either an assay (.xsy) or a chip data file (.xad). The type of assay you have to select depends on the experiment and the Reagent Kit you use to prepare your samples. Details on these assays are described in the Application Notes available for each assay and in the Reagent Kit Guide. 5 Select the desired assay, DNA 1000, for example.
NOTE After a chip run, the results can be evaluated using a different electrophoretic chip data file (.xad) of the same assay type (DNA 1000 in this example). Refer to “Importing Data Analysis Setpoints” on page 259. 6 Select a Destination for the chip data file (.xad) generated as the result of the chip run: 7 Under Data Acquisition Parameters, enter the number of samples you want to be measured. The total number of samples that can be measured varies with the type of assay selected.
Preparing Samples and Chips for Electrophoretic Assays Before you can fill a chip, you have to prepare the samples. To find out how to prepare the samples, refer to the various Reagent Kit Guides available for each LabChip kit. Please refer to these documents for further information and analytical specifications. In general, preparing an electrophoretic assay involves the following steps: • Check that you have everything listed in the appropriate Reagent Kit Guide.
Essential Measurement Practices (Electrophoretic Assays) General: WA R N I N G Wear hand and eye protection and follow good laboratory practices when preparing and handling reagents and samples. WA R N I N G No data is available addressing the mutagenicity or toxicity of the dye/DMSO reagent. Because the dye binds to nucleic acids, it should be treated as a potential mutagen and used with appropriate care.
• Protect dye and gel-dye mix from light. Remove light covers only when pipetting. Dye decomposes when exposed to light. • Use a new syringe and electrode cleaner with each new LabChip kit. • Do not touch the Agilent 2100 bioanalyzer during a chip run and never place it on a vibrating ground. • Keep all reagents and reagent mixes refrigerated at 4 °C when not in use. • Allow all reagents and samples to equilibrate to room temperature for 30 minutes before use. • Use loaded chips within 5 minutes.
Protein Assays: • Store Protein sample buffer at -20 °C upon arrival. Keep the vial in use at 4 °C to avoid freeze-thaw cycles. • Allow the dye concentrate to equilibrate to room temperature for 20 minutes before use, to make sure the DMSO is completely thawed. Protect the dye from light during that time. Vortex before use. • Allow all other reagents to equilibrate to room temperature for 10 minutes before use. • Use 0.5 ml tubes to denature samples.
Loading the Electrophoresis Chip into the Bioanalyzer After preparing the chip, you can insert it into the Agilent 2100 bioanalyzer. To load the chip into the bioanalyzer: 1 Open the lid. NOTE Before inserting the chip, check that the electrode cartridge is installed and the chip selector is in position “1”. For details, refer to “Switching Between Electrophoretic and Flow Cytometric Assays” on page 47. 2 Place the prepared chip into the receptacle. The chip fits only one way. Do not use force.
3 Carefully close the lid. C A U T IO N Do not force the lid closed. This may damage the cartridge. If the lid does not close completely, check that the cartridge and chip are inserted properly, and the chip selector is in the correct position. The icon on the Instrument tab changes to a DNA chip icon, if a DNA assay is selected: If the chip is not detected, open and close the lid again.
Running an Electrophoretic Assay Running an electrophoretic assay in 2100 expert just means pressing a button. NOTE You can stop a chip run at any time, for example, if errors occurred, or if you are not satisfied with the quality of the measurement results, which you can observe during the chip run. See “Stopping a Chip Run” on page 77. Starting the Chip Run When you have loaded the chip, you can start the chip run: 1 On the Instrument tab, click the Start button. The chip run starts.
The number of the sample that is currently being measured is indicated on the information bar: Contents ▲ 73 ▼ Index
The status bar shows the name of the currently measured sample, a progress bar showing the measurement progress for the current sample (not for the whole chip run), and the COM port number used for data acquisition: During the chip run, you can do the following: • View the chip data file in the Data and Assay context by clicking on the name of the Data File: • Switch to any other context.
Finishing the Chip Run When the measurements are finished, the End of run dialog box appears, showing you the number of samples that have been measured, and the file name where the chip run data has been stored. If errors occurred, they would also be displayed in this dialog box. 1 To immediately view the results in the Data and Assay context, you can select the Select file in Data Context check box. 2 Click OK.
The dialog box is closed. • If you selected Select file in Data Context, you are automatically taken to the Data and Assay context, where you can view, analyze, and evaluate the results of your chip run (see “Displaying the Measurement Results (Electrophoresis)” on page 82 and “Analyzing and Evaluating the Results of an Electrophoretic Assay” on page 91).
Stopping a Chip Run You can stop a chip run at any time, for example, • if the quality of the measurement results does not meet your expectations, • if, for example, after three samples you already have the information you desired and you want to start another chip run. NOTE You cannot resume a stopped chip run.
The following message appears: 2 Click Yes to stop the chip run. The End of Run dialog box appears.
The measured samples are marked with a green check, and only these are stored in the chip data file. The unmeasured samples are marked with a white cross on red ground. 3 If you want to immediately view the results in the Data and Assay context, select the Select file in Data Context check box. 4 Click OK. The dialog box is closed.
Entering Chip, Sample, and Study Information During or after a chip run, you can document the run by entering information on chip, samples, and study. 1 In the Data and Assay context, select the Chip Summary tab. 2 On the Sample Information sub-tab, you can enter additional information such as sample names and comments. On the Study Information sub-tab, you can enter the laboratory location, and the name of the experimenter, for example.
NOTE You may find some input fields already filled in, because chip, sample, and study information are taken over from the base assay or chip data file. For details on all input fields, refer to “Chip Summary Tab” on page 427. 3 Click Apply. 4 From the File menu, select Save. TIP You can import chip, sample, and study information from .txt or .csv files. This is especially helpful and time-saving, if you already have documented a similar chip run in another chip data file.
Displaying the Measurement Results (Electrophoresis) You can view the measurement results of an electrophoretic chip run as electropherograms or gel-like images. • You can display the electropherograms either one sample at a time, or all samples at the same time to get an overview of the chip run, for example, to see the progress of a reaction. See “How to Switch Between Single View and Grid View (Electropherograms)” on page 83. • You can navigate through the samples.
How to Switch Between Single View and Grid View (Electropherograms) To switch between single view and grid view: 1 From the Electropherogram menu, select View Single Sample or View All Samples. – OR – Click the View Single Sample Electropherogram tool bar. or View All Samples button on the – OR – Click the All Samples entry in the Tree View Panel to switch to the grid view, or any sample name to switch to the single view.
How to Navigate Through the Samples At any time—even during a chip run—you can scroll through all samples—either in electropherogram or gel view. To navigate through samples using the Tree View Panel: 1 If the tree view is not visible, select View > Tree View. The tree view panel appears to the left of the tabs, and shows all chip data and assay files as nodes. 2 Click any sample name.
To browse through samples: 1 From the Electropherogram or Gel menu, select Next Sample or Previous Sample. – OR – Click the Next Sample or Previous Sample button in the tool bar. To switch between electropherogram and gel view: 1 Click the Electropherogram or Gel tab to display the results of the selected sample as an electropherogram or as a gel-like image. How to Change the Display of Electropherograms and Gel-like Images It is possible to change the display of electropherograms and gel-like images.
To zoom into an electropherogram: 1 From the Electropherogram menu, select Graph Mode > Zoom (default setting). 2 Position the mouse pointer in the electropherogram. 3 Click and hold down the left mouse button. The mouse pointer changes its shape to a magnifying glass . 4 Drag the mouse. A rectangle shows the part of the an electropherogram to be enlarged. 5 Release the mouse button.
To pan and scale an electropherogram: 1 From the Electropherogram menu, select Graph Mode > Pan or Scale. 2 Position the mouse pointer in the electropherogram. 3 Click and hold down the left mouse button. The mouse pointer changes its shape to a double-arrow or to a double crosshair. 4 Drag the mouse. As you drag the mouse, the electropherogram curve moves in the drag direction (Pan mode), or the scales of the X and/or Y axes change (Scale mode). 5 Release the mouse button.
To display data points in an electropherogram: 1 From the Electropherogram menu, select Show Data Points or click the toolbar. button in the Data points used to generate the graph are now shown as bullets. Data points are 0.05 seconds apart. To put a color gradient on the background of an electropherogram: 1 From the Electropherogram menu, select Show Gradient. A color gradient (gray to white) appears on the background of the graph.
Cleaning the Electrodes after an Electrophoretic Assay When the assay is complete, remove the used chip from the bioanalyzer and dispose of it according to the guidelines established by your laboratory safety officer. Remove the chip quickly to prevent a buildup of residues from the solutions on the electrodes. Then perform the cleaning procedure to ensure that the electrodes are clean (i.e., no residues left from the previous assay).
C A U T IO N Never use a cloth to clean the electrodes. Electrostatic discharge could damage the high-voltage power supplies. C A U T IO N Wet electrodes can cause severe damage to the on-board high voltage power supplies. Always make sure the electrodes are dry before inserting them into the bioanalyzer again.
Analyzing and Evaluating the Results of an Electrophoretic Assay The purpose of electrophoretic assays is to calculate the size and concentration of nucleic acid fragments. Results for a particular sample are calculated after all data for that sample has been read.
Data Analysis: DNA The data analysis process for DNA assays consists of the following steps: 1. Raw data is read and stored by the system for all of the individual samples. 2. The data is filtered and the resulting electropherograms of all samples are plotted. You can change the settings of the data analysis after the run and reanalyze your data. 3. Peaks are identified for all samples and are tabulated by peak ID.
4. A sizing ladder (see the example electropherogram below), which is a mixture of DNA fragments of known sizes, is run first from the ladder well. The concentrations and sizes of the individual base pairs are preset in the assay and cannot be changed.
5. A standard curve of migration time versus DNA size is plotted from the DNA sizing ladder by interpolation between the individual DNA fragment size/migration points. The standard curve derived from the data of the ladder well should resemble the one shown below.
6. Two DNA fragments are run with each of the samples, bracketing the DNA sizing range. The “lower marker” and “upper marker” are internal standards used to align the ladder data with data from the sample wells. The figure below shows an example of assigned marker peaks in a sample well. Lower marker Upper marker NOTE The software performs alignment by default. Turning automatic data analysis off (see “Electropherogram Menu” on page 345) suspends data analysis until you turn it on again. 7.
8. To calculate the concentration of the individual DNA fragments in all sample wells, the upper marker, in conjunction with an assay-specific concentration against base-pair size calibration curve, is applied to the individual sample peaks in all sample wells. NOTE The software allows you to redefine the peaks chosen as upper and lower markers. A change in marker selection will cause quantitative changes in the calibration procedure, however, and therefore in the entire data evaluation. 9.
Data Analysis: RNA and Cy5-Labeled Nucleic Acids The data analysis process for RNA and the Cy5-labeled nucleic acids assays consists of the following steps: 1. Raw data is read and stored by the system for all of the individual samples. 2. The data is filtered and the resulting electropherograms of all samples are plotted. You can change the settings of the data analysis after the run and reanalyze your data. 3. Fragments are identified for all samples and tabulated by peak ID.
NOTE Peak ratios for the RNA ladder may vary from one batch of RNA 6000 ladder to the next. Assay performance will not be affected by this variation. 5. For the Eukaryote or Prokaryote Total RNA assay, the RNA fragments (either 18S and 28S for eukaryotic RNA or 16S and 23S for prokaryotic RNA) are detected. After detection, the ratio of the fragment areas is calculated and displayed. 6. To calculate the concentration of the RNA, the area under the entire RNA electropherogram is determined.
RNA Assays The marker solution that is part of each RNA LabChip kit, contains a 50 bp DNA fragment. This fragment is used as lower marker to align all samples. By default the RNA alignment and the subtraction of the lower marker are enabled for RNA Nano assays.
Data Analysis: Protein The data analysis process for protein assays consists of the following steps: 1. Raw data is read and stored by the system for all of the individual samples. 2. The data is filtered and the resulting electropherograms of all samples are plotted. You can change the settings of the data analysis after the run and reanalyze your data. 3. Peaks are identified for all samples and are tabulated by peak ID.
4. A sizing ladder (see the example electropherogram below), which is a mixture of proteins of different known sizes, is run first from the ladder well. The concentrations and sizes of the individual proteins are preset as kDa in the assay and cannot be changed.
5. A standard curve of migration time versus size is plotted from the sizing ladder by interpolation between the individual protein size/migration points. The standard curve derived from the data of the ladder well should resemble the one shown below.
6. Two proteins are run with each of the samples, bracketing the sizing range. The “lower marker” and “upper marker” proteins are internal standards used to align the ladder data with data from the sample wells. The figure below shows an example of assigned marker peaks in a sample well. Lower marker Upper marker NOTE The software performs alignment by default. Turning automatic data analysis off (see “Electropherogram Menu” on page 345) suspends analysis until you turn it on again.
7. The standard curve, in conjunction with the markers, is used to calculate protein sizes for each sample from the migration times measured. 8. To calculate the concentration of the individual proteins in all sample wells, the upper marker, in conjunction with an assay-specific concentration calibration curve, is applied to the individual sample peaks in all sample wells. NOTE The software allows you to define upper and lower markers yourself.
Smear Analysis The 2100 expert software allows to perform a smear analysis for all electrophoresis assays. When the smear analysis is enabled, the software allows you to define regions of interest. These regions are used to define the area of broad peaks and determine their part of the total area. Smear analysis provide a means to analyze broad signals that can be hardly evaluated with the normal peak assignment.
4 Under Smear Analysis select the checkbox Perform Smear Analysis. 5 Click the Apply button that changes become effective.
Performing Smear Analysis After enabling the smear analysis in the setpoint explorer, you are able to insert regions of interest in the electropherogram. To do so: 1 Select the Region Table sub-tab in the Electropherogram tab. 2 Right-click the electropherogram and select Add region. A region will be inserted into the electropherogram. The Region Table will show values for the inserted region.
3 Repeat the previous step until the number of required regions are inserted. 4 Adjust the regions by directly moving the dashed lines in the electropherogram. 5 To remove a region, right-click the dashed line in the electropherogram and select Remove Region from the context menu.
NOTE The smear analysis table can be directly edited by selecting the region table under Smear Analysis in the setpoint explorer.
In the smear analysis table, you can edit the Region Start Size and Region End Size, for example: Contents ▲ 110 ▼ Index
Changing the Data Analysis Different sets of parameters (data analysis setpoints) can be changed in the software in order to modify the data evaluation for sample analysis. For all assays: • Filtering parameters • Peak find parameters for all samples/peak height for individual samples • Enabling smear analysis • Align to upper and/or lower marker For RNA assays only: • Adding/deleting ribosomal fragments These settings can be made before a new run is started or when reanalyzing a previously saved data file.
About the Setpoint Explorer The tool allowing you to modify the data analysis setpoints is the Setpoint Explorer.
To show the setpoint explorer, on the Electropherogram/Gel tab, click the vertical bar on the right edge of the application window: The setpoint explorer appears. For electrophoretic assays, you can modify the setpoints • either globally, that is, for all samples (Global tab), • locally, for the current sample (Local tab). Click the + nodes to expand, and the – nodes to collapse branches. Setpoints that you can change are white.
Changing any global setpoints while local settings have been applied opens a dialog box asking whether to override custom sample settings: Choosing Yes causes any changes made to the setpoints for individual samples to be discarded and applies the changes globally to all samples. Choosing No causes individual samples to retain changed setpoints.
Filtering Setpoints The first step the software takes in analyzing the raw data is to apply data filtering. The following filtering setpoints can be changed: Filter Width Defines the data window, given in seconds, used for averaging. The broader the filter width, the more raw data points are used for averaging. As a result, the noise level will decrease, but peaks will become lower and broader.
The four peak find setpoints that can be changed are: Min Peak Height The Min Peak Height setpoint determines whether a peak is kept. For each peak, the difference between the start point value and the center point value (local baseline) must be greater than the Min Peak Height value. Min Peak Width The Min Peak Width setpoint determines the minimum amount of time that must elapse before a peak is recognized.
Manually Moving Fragment Start and End Points (RNA and Cy5-Labeled Nucleic Acids) It is also possible to alter the start and end points manually for individual fragments in an RNA or Cy5-labeled nucleic acids assay. Zooming in on the base of a particular fragment allows you to see the start and end points. Placing the cursor over one of these points changes the cursor to a pointing hand, allowing you to click and drag the point along the line of the fragment until it is positioned as desired.
TIP The fragment table can be directly edited in the setpoint explorer: NOTE Changing the start or end points of the fragment will change the calculated rRNA ratio. It might be convenient to pause the automatic analysis (Electropherogram > Pause Automatic Analysis) until all changes are done.
Setting the Baseline for Calculation of RNA Concentration At low signal-to-noise ratios, the baseline that defines the area used for calculating the concentration of RNA assays is highly dependent on the settings for the Start and End Time. You can adjust the Start and End Times manually (thereby adjusting the baseline) to ensure a good result even at very low signal-to-noise ratios. Choose a single sample.
Assigning Upper and Lower Marker Peaks For each DNA or protein sample, the upper and lower marker peaks are assigned first and then the data is aligned so that the sample markers match the ladder markers in time, allowing the size and concentration of the sample peaks to be determined. The first peak is assigned to be the lower marker and is then offset to match the lower marker in the ladder.
Aligning or Unaligning the Marker Peaks (DNA or Protein assays only) The upper and lower are then aligned to the ladder markers by resampling the sample data in a linear stretch or compression using a point-to-point fit: Data before alignment Contents ▲ 121 ▼ Index
Markers aligned to the ladder If the sample marker peaks are either more than twice as far apart or less than half as far apart as the ladder markers, they are assumed to be the wrong peaks, and analysis of the sample stops, producing the error “Marker peaks not detected”.
NOTE With DNA and protein assays, the height of marker peaks is assay dependent. Ladder peaks are analyzed to calculate a marker peak threshold that is used to locate the marker peaks in the sample wells. If the marker peaks found using this calculated method fail to align with those of a sample, the 2100 expert software will use the minimum peak height threshold setting instead (if this value is lower than the value for the marker peak).
Manual Integration For all electrophoretic assays, the 2100 expert software allows to manually integrate peaks. Manual integration allows you to move, add or delete peak baselines. TIP To move a peak baseline, point along the vertical line, press the CTRL key and left mouse button. To move a peak baseline, point along the signal, press the left mouse button only.
The baseline points become visible as blue or green dots. Highlighted baseline points are labelled green and can be moved either along the vertical line (press CTRL key and left mouse button) or along the signal trace (left mouse button). The blue baseline points are fixed and cannot be moved. To highlight a baseline point, click on it. 3 Pause the automatic analysis by clicking the appropriate icon in the toolbar.
4 Adjust the baseline points as appropriate. TIP To move a peak baseline point along the vertical line, press the CTRL key and the left mouse button. To move a peak baseline point along the signal, press the left mouse button only. 5 Click the Automatic Analysis button to enable the integration again. The integration results in the result and peak tables will change according to the changes done.
Example: Removing peaks To remove peaks: 1 Highlight the Electropherogram tab in the Data and Assay context and zoom into the electropherogram to enlarge the peak of interest. 2 Select Electropherogram > Manual Integration to switch off the automatic integration. As an alternative you might click the Manual Integration button in the toolbar. The baseline points become visible as blue or green dots.
3 Right-click on a baseline-point and select Remove Peak from the context menu.
Example: Inserting peak baselines To insert peaks manually: 1 Highlight the Electropherogram tab in the Data and Assay context and zoom into the electropherogram to enlarge the peak of interest.
2 Right-click on the electropherogram and select Add Peak from the context menu.
3 Two baseline points and the connecting line will appear and the integration results shown in the result and peak tables will be updated. 4 Pause the automatic analysis by clicking the appropriate icon Contents ▲ 131 ▼ in the toolbar.
TIP Before manual integration is done, it is strongly recommended to pause the automatic analysis and to restart it again when all changes are done. Otherwise data analysis starts every time, when the baseline points are moved. 5 Adjust the baseline points as appropriate. TIP To move a peak baseline point along the vertical line, press the CTRL key and the left mouse button. To move a peak baseline point along the signal, press the left mouse button only.
6 Click the Automatic Analysis button to enable the integration again. The integration results in the result and peak tables will change according to the changes done.
Reanalyzing a Chip Data File NOTE Occasionally you may wish to open and view or reanalyze a chip data file that was run and saved previously. The raw data values are saved in the data file, along with the analysis settings that were chosen for the run, so that the data can be reanalyzed with different settings. To do this: 1 Click File > Open... to open a chip data file (.xad). 2. Choose the filename from the list of data files. 3. Click OK.
RNA Assays Only: • Fragment names and colors associated with labels • Fragment start/end times, additional fragments (or delete fragments found) DNA and Protein Assays Only: • Exclude peaks from analysis • Reassign upper/lower markers • Alignment or no alignment with ladder peaks • Assay (you can save the changed settings under a new assay name, if desired) TIP When applying modified data analysis setpoints, the software will (by default) immediately recalculate the raw data, which takes some time.
Comparing Samples from Different Electrophoretic Chip Runs The 2100 expert software allows you to compare the measurement results of samples from different electrophoretic chip runs. Samples to be compared must be from chip runs of the same assay type. In the Comparison context, you can create comparison files, include samples from different chip runs, and compare the samples by overlaying electropherograms, for example.
The .xad files appear in the Select Data Files list of the Tree View Panel. NOTE The Select Data Files list also contains all electrophoretic .xad files that are open in the Data and Assay context. 3 Select a .xad file from the Select Data Files list to display a list of its samples. 4 Right-click a sample name and select Add Sample to New Comparison File.
A new comparison file appears in the upper part of the tree view containing the sample. The sample is selected and its electropherogram is shown. Note that the Electropherogram Tab (Single/Grid View) a has the same functionality as in the Data and Assay context.
5 You can now add further samples from any of the open .xad files to the comparison file. TIP Double-clicking a sample name in the lower part of the tree view or dragging a sample name into the tree view adds the sample to the comparison file that is currently selected in the upper part of the tree view. Or, if no comparison file is selected, creates a new comparison file and adds the sample to it.
The following message appears if you try to add a sample of a .xad file which has the wrong assay type: 6 You can also remove samples from a comparison file. Right-click the sample name and select Delete Sample from Comparison File.
7 When you have added all your samples you can select the Comparison Summary Tab which displays information on the comparison file, and lets you enter a comment regarding the comparison.
8 To compare the electropherograms of samples, go to the Electropherogram tab, click Overlaid Samples in the toolbar (see “Data and Assay Context – Electrophoresis Toolbar” on page 369), and select the samples to be compared.
9 Select the Gel tab to display a comparison of the gel-like images of the samples. Note that the Gel Tab has the same functionality as in the Data and Assay context. 10From the File menu, select Save to save the comparison file (.xac) under the default name, or select Save As... to save it under a new name. The default name is derived from the assay class: “ComparisonFileX [Assay Class].xac” where “X” is an autoincremented number. Example: “ComparisonFile0 Protein 200.
NOTE You can re-open comparison files to review the comparison results, and to add/remove samples.
Result Flagging Result flagging can be used to assign a user-defined color code to a sample. This lets you easily identify samples with certain properties immediately after a chip run. The color assignment is carried out by applying a sequence of rules to the measurement results obtained for the sample. The rules are defined on chip level and are applied to all samples of the chip.
TIP The examples shown in this chapter are taken from the demo assay “Demo Protein 200 Plus.xsy”, that comes with the 2100 expert software. You can find this demo assay in the “..\assays\demo\electrophoresis” subdirectory of the 2100 expert installation folder. In the “..\data\samples\resultflagging” subdirectory of the 2100 expert installation folder, you can find further examples for result flagging rules (.xml) which you can import in the “Protein 200 Plus” demo assay.
Defining Result Flagging Rules The rules can be defined on the Result Flagging tab, which is available in the Data and Assay context if an electrophoretic chip data (.xad.) or assay (.xsy) file is selected.
A result flagging rule consists of the following: • Label Expression An optional description for the rule used to label samples meeting the rule. • If Expression An expression built from predefined functions, variables, and logical operators. • Comment An optional comment for the rule. • Color Expression A solid color or a color gradient built from two colors, used for flagging samples that meet the rule. Details on all elements of the Result Flagging tab are given in “Result Flagging Tab” on page 474.
Color Indication Result flagging shows: • On the Chip Summary Tab: The colors in the Result Flagging column show which sample matches which rule.
• On the Gel Tab: The spot on top of the lane is colored if the sample matches a result flagging rule.
• On the Results tab: Result Flagging Color: color of the result flagging rule that the current sample matches. Result Flagging Label: label of the result flagging rule that the current sample matches. How to Define Result Flagging Rules Two modes are available to define result flagging rules: • Assisted Mode In this mode you can easily compose an expression by selecting functions and operators from given lists. If necessary, additional attributes have to be provided.
TIP You can export result flagging rules and import rules from other assay or chip data files. See “Exporting Result Flagging Rules” on page 274 and “Importing Result Flagging Rules” on page 262. To define a result flagging rule for an electrophoretic assay: 1 In the Data and Assay context open and select an electrophoretic chip data (.xad) or assay (.xsy) file. 2 Switch to the Result Flagging tab. 3 Create a new rule by clicking the Add Rule button in the Result Flagging toolbar.
6 Enter an expression in the If Expression box. The expressions can be composed by making selections in the drop-down lists on the right and entering values. Multiple expressions can be combined with the logical operators AND, AND NOT, OR, OR NOT. Refer to “Result Flagging Tab” on page 474 for available functions, operators, and variables.
NOTE Please refer to the function reference in the Help Pane (which appears when you select a function from the list) for details on syntax, usage and examples. 7 Under Color Expression, define a color for the rule. All samples that meet the rule will be highlighted with this color (see “Color Indication” on page 149). If the Gradient checkbox is enabled you can assign a color gradient to each rule (for example, to display differences in concentration).
Example: Result Flagging Sample 1 contains 100 ng/µl proteins. The electropherogram shows 2 peaks for 2 different proteins, which could be separated. One peak can be found at 32 kDa (LDH). Sample 2 contains 60 ng/µl proteins and shows 3 peaks. Sample 3 contains 80 ng/µl proteins and shows 5 peaks. Now, the following rules are defined (the syntax is explained in “Result Flagging Tab” on page 474): 1. Is there a peak at 30 kDa +/- 7%? Rule 1: PeakFound(30, PER, 7) 2.
Running and Evaluating Flow Cytometric Assays For running and evaluating flow cytometric assays, you need to know the following: • “Principles of Flow Cytometric Measurements” on page 157 • “Overview of Flow Cytometric Assays” on page 166 • “Preparing and Running a Flow Cytometric Assay” on page 169 • “Analyzing and Evaluating the Results of a Flow Cytometric Assay” on page 198 Contents ▲ 156 ▼ Index
Principles of Flow Cytometric Measurements Besides electrophoretic assays (DNA, RNA, and proteins), the Agilent 2100 bioanalyzer supports flow cytometric assays: • First, cells are stained with two fluorescent dyes that correspond to biologically relevant parameters, as described in the application notes available for each assay. “Staining Cells” on page 158 explains the principle. • Next, the stained cells are analyzed on the chip.
Staining Cells With the 2100 expert software, you can differentiate several properties of a cell. The characteristics that are examined depend on the dye, which binds specifically to a cellular constituent or is metabolized by the cell to generate a fluorescent product. You usually use two dyes with different colors. Typically, one of the two dyes is used as reference dye to select the target cells (living/dead, cell line type, ...). The second dye can be used to detect another characteristic of the cell.
The following dyes are recommended for use as the red stain: Dye (red fluorescence) Max. Excitation wavelength Max.
Cell Detection with the Agilent 2100 Bioanalyzer LabChip technology allows cell measurements by integrating cell flow, hydrodynamic focusing, and fluorescence detection into a microfluidic chip. A cell suspension can be confined or “pinched” to a portion of a microfluidic channel, causing cells to line up in single file for individual cell detection. The following images illustrate the pinching process.
Measuring Events The bioanalyzer counts cells stained with fluorescent dyes and measures their fluorescence intensities. Each cell or bead that passes the detector and emits fluorescence above a threshold value is counted as an event. For each event, the intensity of two different fluorescent signals (red and blue) is recorded. The intensity of the fluorescent signal depends on the amount of stain bound to the cell (and therefore a specific cell property) and the physical properties of the stain itself.
Generating Histograms 2100 expert counts the events, sorts them and displays them according to their fluorescence intensity in histograms. For each color measured, a histogram displays the number of events related to the fluorescence intensity. A large number of events with a high fluorescence value means that a large number of cells containing the fluorescence dye were detected. In the following illustration, cells which fluoresce in both colors are highlighted.
In the histograms, the bar chart is replaced by a point-to-point line as shown in the following image. For detailed information, see “Using Histograms for Evaluation” on page 199.
Generating Dot Plots Single events can also be displayed related to both fluorescence values, generating a map of dot plots. In dot plot view, the events (cells with a minimum fluorescence intensity) are displayed in a coordinate system (logarithmic axis scaling). Each axis represents a fluorescence color. A high number of events (cells) with similar fluorescence values are displayed as a dense cluster of dots, as shown in the following image.
In predefined assays, the borders of the rectangular region represent the markers defined in the corresponding blue and red histograms. Gate The lower left region of a dot plot usually shows no events, due to the defined peak detection threshold that the fluorescence values must exceed. For detailed information, see “Using Dot Plots for Evaluation” on page 220.
Overview of Flow Cytometric Assays The cell characteristic to be measured requires not only specific dyes. Several measurement parameters to control the measurement and the data acquisition parameters also have to be specified. These so-called “setpoints” are stored in assay files (.xsy) and are read by the 2100 expert software before it starts the measurement.
• Red to blue This assay is for applications that apply a red reference dye and analyze blue fluorescent cells within a red population. • Checkout Beads Red checkout beads are loaded into the wells 1, 3 and 5 and blue checkout beads into the wells 2, 4 and 6. Markers are set according to expected fluorescence levels of the red and blue beads. The Checkout Beads assay has the properties of a generic assay (see below).
The regions of the dot plots are related to the markers of the histograms. Thus, the results of the dot plots are identical to the results of the histograms. Generic assay This assay has no specific settings and can be used to define individual assays. You can freely add markers or regions, and define the gating direction. The generic assay is recommended for chips with different samples and stainings, where regions would need to be defined individually.
Preparing and Running a Flow Cytometric Assay A flow cytometric chip run requires the following steps: 1. Set up and switch on the Agilent 2100 bioanalyzer. Refer to “Switching Between Electrophoretic and Flow Cytometric Assays” on page 47. 2. Start the 2100 expert software. Details are given in “Starting 2100 Expert” on page 32. 3. Select a flow cytometric assay. See “Selecting a Flow Cytometric Assay for a Chip Run” on page 171. 4. Prepare chip and samples.
• Analyze and evaluate the results: – “Using Histograms for Evaluation” on page 199 – “Using Dot Plots for Evaluation” on page 220 – “Evaluating Antibody Staining, Apoptosis, and GFP Assays” on page 229 Contents ▲ 170 ▼ Index
Selecting a Flow Cytometric Assay for a Chip Run To select an assay: 1 Switch to the Instrument context. 2 In the Tree View Panel, select the bioanalyzer you want to use. In the upper left of the Instrument tab, an icon shows the status of the bioanalyzer.
3 If you do not see one of these icons, check that the bioanalyzer is switched on and properly connected: – Check the COM port setting. – Make sure the bioanalyzer is physically connected to the PC (over the serial interface). – Check the power connection. – Check the power switch. If you need additional help, please refer to the Agilent 2100 Bioanalyzer Maintenance and Troubleshooting Guide. 4 Select an assay for the chip run. On the Instrument tab, click the Assay... button. – OR – Click the Assays menu.
Both will open the Assays menu, allowing you to select an assay from the submenus. – OR – Select File > Open File to Run.... This opens a dialog box, allowing you to load either an assay (.xsy) or a chip data file (.xad). The type of assay you have to select depends on the experiment and the staining protocol you use to prepare your cell samples. Details on these assays are described in the Application Notes available for each assay. 5 Select the desired assay, Apoptosis, for example.
NOTE After a chip run, the results can be evaluated using a different flow cytometric chip data or assay file. Refer to “Importing Data Analysis Setpoints” on page 259. 6 Select a Destination for the chip data file (.xad) generated as the result of the chip run.
a Enter the number of samples you want to be measured. When preparing the chip (see “Preparing Samples and Chips for Flow Cytometric Assays” on page 176), keep in mind that you have to follow the sequence of the sample wells. For example, if you want to measure 3 samples, you have to fill the wells 1, 2, and 3 with your samples, and the remaining wells with cell buffer solution. b Select the Data Acquisition Mode.
Preparing Samples and Chips for Flow Cytometric Assays WA R N I N G Several substances such as dyes can have toxic, carcinogenic, or mutagenic potential. Therefore, carefully follow the safety instructions from the dye safety data sheet and the Reagent Kit Guides. Also read the “Essential Measurement Practices (Flow Cytometric Assays)” on page 178. Before you can fill a chip, you have to prepare the samples.
Chip Reagents Several reagents have to be added to the chip to prepare it for measurement. The following image shows which reagents have to be filled in which wells. Priming solution Cell buffer Sample 1 – 6 Cell buffer Focusing dye solution Make sure you follow these directions when preparing the sample: • The priming solution has to be added first. It fills all channels (removes all air from the micro channels). • The focusing dye is used to adjust the optic.
Essential Measurement Practices (Flow Cytometric Assays) • Handle and store all reagents according to the instructions given in the Reagent Kit Guides. • Avoid sources of dust or other contaminants. Foreign matter in reagents and samples or in the wells of the chip will interfere with assay results. • Store all reagent and reagent mixes in the dark and refrigerated at 4 °C when not in use. • Allow all reagents to equilibrate to room temperature for 30 minutes before use. • Protect focusing dye from light.
• Before bead preparation, vortex bead vials for 15 seconds. • Prepared chips must be used within 5 minutes. If a chip is not run within 5 minutes, beads may settle or reagents may evaporate, leading to poor results. • Never touch the instrument lens. • Never touch the Agilent 2100 bioanalyzer during a chip run and never place it on a vibrating ground.
Loading the Cell Chip into the Bioanalyzer After preparing the chip, you can insert it into the Agilent 2100 bioanalyzer. NOTE Before inserting the chip, check that the pressure cartridge is installed and the chip selector is in position “2”. For details, refer to “Switching Between Electrophoretic and Flow Cytometric Assays” on page 47. To load the chip into the Agilent 2100 bioanalyzer: 1 Open the lid. 2 Place the prepared chip into the receptacle. The chip fits only one way. Do not use force.
3 Carefully close the lid. C A U T IO N Do not force the lid closed. This may damage the pressure cartridge. The lid may not close completely. If the software recognizes that a chip has been inserted, the system is ready. If the chip is not recognized open the lid, verify that the cartridge and chip are inserted properly and the chip selector is in the correct position. Close the lid. The adapter with the gasket in the cartridge fits onto the priming well of the chip.
Running a Flow Cytometric Assay Running a flow cytometric assay in 2100 expert just means pressing a button. NOTE You can stop a chip run at any time, for example, if errors occurred, or if you are not satisfied with the quality of the measurement results, which you can observe during the chip run. See “Stopping a Chip Run” on page 187. Starting the Chip Run When you have loaded the chip, you can start the chip run: 1 On the Instrument tab, click the Start button.
The Dot Plot sub-tab shows single events (cells) as they are detected, displayed as dots. In the coordinate system, the red and blue fluorescence intensity of each event can be read. The name of the currently measured sample is displayed above the graph.
The status bar shows the name of the currently measured sample, a progress bar showing the measurement progress for the current sample (not for the whole chip run), and the COM port number used for acquiring data: During the chip run, you can do the following: • View the chip data file in the Data and Assay context by clicking on the name of the Data File: • Evaluate any chip data file in the Data and Assay context. • Compare samples in the Comparison context.
Finishing the Chip Run When the measurements are finished, the End of run dialog box appears, showing you the number of samples that have been measured, and the file name where the chip run data has been stored. If errors occurred, they would also be displayed in this dialog box. 1 To immediately view the results in the Data and Assay context, you can select the Select file in Data Context check box. 2 Click OK.
The dialog box is closed. • If you selected Select file in Data Context, you are automatically taken to the Data and Assay context, where you can view, analyze, and evaluate the results of your chip run (see “Displaying the Measurement Results (Flow Cytometry)” on page 192 and “Analyzing and Evaluating the Results of a Flow Cytometric Assay” on page 198).
Stopping a Chip Run You can stop a chip run at any time, for example, • if the quality of the measurement results does not meet your expectations, • if, for example, after three samples you already have the information you desired and you want to start another chip run. NOTE You cannot resume a stopped chip run.
The following message appears: 2 Click Yes to stop the chip run. The End of Run dialog box appears. The measured samples are marked with a green check, and only these are stored in the chip data file. The unmeasured samples are marked with a white cross on red ground.
3 If you want to immediately view the results in the Data and Assay context, select the Select file in Data Context check box. 4 Click OK. The dialog box is closed. • If you selected Select file in Data Context, you are automatically taken to the Data and Assay context, where you can view, analyze, and evaluate the results (if any) of your chip run (see “Displaying the Measurement Results (Flow Cytometry)” on page 192 and “Analyzing and Evaluating the Results of a Flow Cytometric Assay” on page 198).
Entering Chip, Sample, and Study Information During or after a chip run, you can document the run by entering information on chip, samples, and study. 1 In the Data and Assay context, select the Chip Summary tab. 2 On the Sample Information sub-tab, you can enter additional information for samples, such as names for blue and red stain. On the Study Information sub-tab, you can enter the laboratory location, and the name of the experimenter, for example.
NOTE You may find some input fields already filled in, because chip, sample, and study information are taken over from the base assay or chip data file. For details on all input fields, refer to “Chip Summary Tab” on page 427. 3 Click Apply. 4 From the File menu, select Save. TIP You can import chip, sample, and study information from .txt or .csv files. This is especially helpful and time-saving, if you already have documented a similar chip run in another chip data file.
Displaying the Measurement Results (Flow Cytometry) You can view the measurement results of a flow cytometric chip run as histograms or dot plots. • You can display the histograms/dot plots either one sample at a time, or all samples at the same time to get an overview of the chip run, for example, to see the progress of a reaction. See “How to Switch Between Single View and Grid View” on page 193. • You can navigate through the samples. See “How to Navigate Through the Samples” on page 194.
How to Switch Between Single View and Grid View To switch between single view and grid view: 1 From the Histogram or Dot Plot menu, select Single View or Grid View. – OR – Click the Single View or Grid View button on the histogram/dot plot tool bar. – OR – Click the All Samples entry in the Tree View Panel to switch to the grid view, or any sample to switch to the single view. – OR – Double-click any histogram or dot plot in the grid view to switch to single view.
How to Navigate Through the Samples At any time—even during a chip run—you can scroll though all samples—either in histogram or dot plot view. To navigate through samples using the Tree View Panel: 1 If the tree view is not visible, select View > Tree View. The tree view panel appears to the left of the tabs, and shows all chip data and assay files as nodes. 2 Click any sample name. The histogram or dot plot of the sample is shown in single view.
How to Change the Display of Histograms and Dot Plots In single view, it is possible to change the display of histograms and dot plots. In histograms and dot plots you can: • zoom (enlarge or reduce using the mouse) the graphs to display details, for example. • put a color gradient on the background of the graphs. In histograms, you can additionally: • show data points. To zoom into a histogram or dot plot: 1 Position the mouse pointer in the histogram/dot plot. 2 Click and hold down the left mouse button.
You can perform several zoom steps in a row. When you have zoomed a histogram or dot plot, the Undo Zoom and Undo All buttons are enabled. To undo one zoom step: 1 Click the Undo Zoom button or double-click in the histogram or dot plot. To undo all zoom steps: 1 Click the Undo Zoom All Contents button.
To display data points in histograms: 1 From the Histogram menu, select Show Data Points. All events are shown as bullets. To put a color gradient on the background of a histogram or dot plot: 1 From the Histogram or Dot Plot menu, select Gradient. – OR – Click the Gradient button the histogram or dot plot toolbar. A color gradient (gray to white) appears on the background of the graph.
Analyzing and Evaluating the Results of a Flow Cytometric Assay You can analyze and evaluate result data of flow cytometric assays using either the dot plot or the histogram view. In both views, you can evaluate the detected cells by defining areas of interest. • Histograms show the distribution of events related to the red and blue fluorescence intensity. Gating is used to generate subsets based on markers in one histogram. See “Using Histograms for Evaluation” on page 199 for detailed information.
Using Histograms for Evaluation Histograms are graphical representations of the measurement results, where the number of events (cells) is mapped to the Y axis and their fluorescence values to the X axis. The resulting curves show the frequency distribution of the events in relation to their fluorescence intensity values, as shown in the following image. In real histograms, the bin is reduced to a dot (data point).
Markers Markers are used to define a range of fluorescence intensity values in a histogram. The upper and lower limits of the range are displayed as vertical lines, as shown in the following image. Lower limit of the marker Upper limit of the marker Events (cells) of interest Low intensity The numerical values for each defined marker are displayed in a separate row in the result table. One marker is used as a gate for the second histogram, to define a subset of events.
Gating Gating is used to restrict the number of events that are evaluated by gating out (filtering) events that do not have the fluorescence values set by a marker. For example, by gating on a blue marker, you can exclude all events with low blue fluorescence (allowing you, for example, to gate out dead cells, unbound dye and debris). Only events with blue fluorescence values within the marker range are evaluated.
• Gating from red to blue uses the red histogram to define the subset by a marker (GFP assay). NOTE Predefined assays have a fixed gating direction, while assays of type Generic have a variable gating direction.
The following figures illustrate gating from blue to red. The two histograms display all measured events in the blue histogram and in the red histogram without gating. In this case, you cannot see which cells fluoresce only in the blue and which fluoresce only in the red.
By setting a marker on the blue histogram, you can define the blue fluorescence range that must be met for a cell to be considered for the red histogram. You use the gating on the blue histogram to define a subset for the red histogram.
The red histogram displays now only cells with blue and red fluorescence within the marker. To evaluate this subset, you can set a marker in the red histogram. This second marker filters out all cells that do not have fluorescence in this range.
The following image shows two histograms with a gating direction from blue to red (left to right) of an apoptosis assay. The blue histogram shows calcein fluorescence, which indicates living or dead cells (high fluorescence value means living cells). The red histogram shows the subpopulation of living cells with annexin V fluorescence indicating apoptosis (high fluorescence value means the cell is apoptotic). As a result you can see a subset of living, apoptotic cells.
How to Insert a Marker in a Histogram A marker is shown as two vertical lines that define a region of fluorescence values. It is used to select a subset of events according to this fluorescence region. NOTE You can insert markers only in generic assays. To add a new marker: 1 In the tool bar of the Histogram tab, click the Insert Marker button . A marker is added to the selected histogram window.
You can remove markers that you do not need any more: 1 Click on one of the vertical lines in the histogram to select the marker. The lines of the selected marker are displayed bold. TIP You can also click the corresponding row in the result table to select the marker. 2 Click the Delete Marker button Contents to remove the marker.
How to Configure Markers You can change the color, name, and the upper and lower limits of the marker: 1 Double-click the desired marker. – OR – Right-click the corresponding row in the result table and select Configure Marker... from the context menu. – OR – Select the marker and click the Configure Marker button in the toolbar. The Configure Marker dialog box appears. 2 Enter a name for the marker, for example, the used dye (it is advisable to use names that identify the marker).
NOTE The lower and upper values must be within the range of 0.01 – 10000 relative fluorescence units. 5 Click the Color button to open the Color dialog box and select a color. 6 Click OK.
How to Move the Upper and Lower Limits of Markers You can change the position of both marker lines by dragging them with the mouse: 1 Position the mouse pointer on a marker line. The mouse pointer changes its shape to a hand. 2 Drag the line to the desired position. 3 Release the mouse button. 4 Repeat these steps for the other marker line, if necessary.
How to Copy Markers to All Histograms Once a marker is defined, you can copy it in the histograms of all samples (generic assays only): 1 Select the marker in the histogram or in the result table. The Insert the selected marker into all histograms button is now enabled. 2 Click this button. The Copy Marker dialog box appears. This dialog box asks you whether or not you want to use the marker as reference. 3 Click Yes to use this marker as reference.
To set the gating direction: 1 Select the marker in the red or blue histogram you want to use as a gate for the other histogram. The corresponding gating button in the tool bar is now enabled. 2 Click or to set the gating direction. – OR – Right-click the marker in the histogram or in the result table, and select Gate in Red/Blue histogram from the context menu. The gating direction is displayed in the Information Bar. If the gating direction is already set, you first have to remove the existing gating.
How to Overlay Histograms You can compare samples by overlaying their gated histograms. This is useful, for example, if you want to see the progress of a reaction or if one sample is used as reference. Overlaying histograms might also be helpful for adjusting the marker position. You can overlay all measured samples. Both red and blue histograms are overlaid.
To overlay all samples: 1 Click the Overlaid Samples button to open a drop-down list. 2 Select All Samples to overlay the histogram curves of all samples. To remove histograms from the overlay: 1 Select the sample that contains the overlaid histograms. 2 Click the Overlaid Sample button to open the drop-down list. 3 Click the sample that you want to be removed. – OR – Click No Overlay to remove all overlaid curves from the histogram.
How to Set Signal Colors for Overlaid Histograms You can use the Graph Settings tab in the Options dialog box to configure the signal colors (colors of curves in histograms): 1 Select Tools > Options. 2 Click the Graph Settings tab to bring it to the front. To configure the signal color: 1 Click the colored square corresponding to the signal. The Color dialog box appears. 2 Select a color for the signal and assign it by clicking OK.
Displaying the Results of Histogram Evaluations The calculated results are displayed in result tables, one table below each histogram. Markers, gates, several statistical values, and the %-values of events are shown in the result tables. Each marker you insert in the histogram gets its own row. Note that you can only use one marker for gating. The additional markers can be used to evaluate different parts of the histogram statistically. If the option Hide superset curve...
The content of the result tables depends on the gating direction. The histogram that is used for gating can display the following results: Marker All events – this row shows the data for all measured events, for example, for all living and dead cells. The following rows show the data for the subset of cells defined by the inserted marker. If you use a predefined assay, the entry can be “Calcein on all events”, for example. Min Minimum fluorescence value of the corresponding marker.
The histogram that displays the gated data can show the following data: Marker All events – this row shows the data for all events that pass the gate. The following rows show the data for all events covered by the inserted marker. If you use a predefined assay, the entry can be “Annexin V on subset”, for example. Min Minimum fluorescence value of the corresponding marker. Max Maximum fluorescence value of the corresponding marker. Events Number of events covered by the marker.
Using Dot Plots for Evaluation On the Dot Plot tab, cells are displayed as dots, where their red fluorescence intensity is mapped on the Y axis and their blue fluorescence intensity is mapped on the X axis. NOTE The lower left region of the dot plot area may show no events, because of the threshold for event detection. Dots are only displayed if their fluorescence intensity exceeds a minimum limit. The limits are specified in the assay—separately for red and blue fluorescence.
How to Add Regions to Dot Plots (Generic Assay only) You can draw regions in dot plots of generic assays. If there are regions already defined in other samples, you can copy these regions in the dot plot of the current sample. To draw a new region: 1 Click the Insert Region button in the toolbar. The mouse pointer changes its shape to a crosshair. 2 Draw a rectangle into the dot plot. New regions are automatically named “Region x”, where x is an auto-incremented number.
To insert an existing region: 1 Select the sample where you want to insert an existing region from another sample and click Insert existing region . The Insert Region dialog box appears. 2 Select the region that you want to insert and click Insert Region. The region is inserted at its predefined position. To remove a region: 1 Click the region border to select the region that you want to remove. The selected region is highlighted. and the Delete Regions button is enabled. 2 Click this button.
How to Configure Regions You can change the color of the region border, edit the region’s name, and define the position and size of the region. To configure a region: 1 Double-click the border of the region that you want to configure. – OR – Right-click the corresponding row in the result table and select Configure Region... from the context menu. – OR – Click the region border to select the region, and click the Configure Region button in the toolbar. The Configure Region dialog box appears.
3 Enter fluorescence values for the left, right, bottom, and top side of the rectangle to define position and size of the region. These values correspond to the upper and lower marker limits of the blue and red histograms. 4 Click the ... button next to the color square to open the Color dialog box, and select a color for the region border. 5 Click OK. To color dots inside regions: 1 Click the Color Dots button in the toolbar. All dots inside the regions now have the same color as the region border.
How to Change Position and Size of a Region You can change the size and position of regions to restrict the number of included events. You can work graphically with the mouse or enter the values in the Configure Region dialog box. To move a region: 1 While pressing the Shift key, click the region border, and drag the region to the new position. 2 Release the mouse button. To change size and position with the mouse: 1 Click the region border to select the region.
To change size and position numerically: 1 Double-click the region to open the Configure Region dialog box. 2 Enter fluorescence values for the left, right, bottom, and top side of the rectangle to define position and size of the region. These values correspond to the upper and lower marker limits of the blue and red histograms. 3 Click OK. How to Insert a Region in All Dot Plots If you have defined a region for one sample, you can copy it to the other samples of the assay.
Click No to create new regions that are not “connected”. The region will be inserted in the dot plots of all other samples. When the properties of the region are changed, the changes affect only the selected sample. The region is copied to all samples of the assay. How to Work with Gates in Dot Plots You can insert gates only in generic assays. For predefined assays, the gate is already defined.
Displaying the Results of Regions The measurement results and calculations for regions are displayed in the result table below the dot plot. In predefined assays, only one region is available, while for generic assays, dot plots can have as many regions as you like. The following values are displayed: Region The first region (All Events) always displays the values for all detected events.
Evaluating Antibody Staining, Apoptosis, and GFP Assays With the 2100 expert software, several predefined assays are supplied. You should only use each assay for the specific experiment for which it was developed. For example, you have to use the read dye for detection of apoptosis (calcein and Cy5, for example): • “Evaluating Antibody Staining Assays” on page 230. • “Evaluating Apoptosis Assays” on page 235. • “Evaluating GFP Assays” on page 240.
Evaluating Antibody Staining Assays Antibody staining lets you measure protein expression on the surface or inside a cell by means of specific antibodies. Either the primary antibody itself is conjugated with a dye or you must use a labeled secondary antibody that recognizes the primary antibody. When you measure the fluorescence of the cells, you can determine the amount of cells with attached antibodies. Typically, you can use a red dye such as APC (Allophycocyanin) or Cy5 to measure antibody presence.
Histogram Evaluation The blue histogram is used for gating. High fluorescence in the blue histogram means that the cells are living (if a life-indicating dye is used). Low fluorescence means the cells are dead. If you use a nucleic acid dye, you cannot distinguish between living and dead cells, you can only count all measured cells.
When using the calcein marker in the blue histogram for gating, only living cells are considered for building the histogram of the red dye. High red fluorescence values indicate living cells with bound antibodies, low red fluorescence values living cells without bound antibodies. See the following example.
Dot plot evaluation If you switch to the Dot Plot tab, one region is displayed in the dot plot. The red fluorescence values of the region are related to the marker in the red histogram, the blue fluorescence values to the marker in the blue histogram. As in the histogram evaluation, high blue fluorescence and high red fluorescence mean living cells with bound antibodies. See the following example.
The results of the dot plot evaluation are numerically displayed in the result table: Events covered by the region All measured events Amount of living cells in relation to all measured cells Amount of living cells with high antibody binding in relation to all living cells Contents ▲ 234 ▼ Index
Evaluating Apoptosis Assays The apoptosis assay can be used to examine how many apoptotic cells are within a living cell population. Dead (or necrotic) cells can be excluded from the evaluation. For a detailed description on how to evaluate the results using histograms and regions, refer to “Using Histograms for Evaluation” on page 199 and “Using Dot Plots for Evaluation” on page 220. Living or dead cells In most cases, you want to know whether cells are dead or alive at a specific time.
Histogram evaluation The two histograms displaying the results of the assay are related to calcein (blue fluorescence) and annexin V (red fluorescence). High fluorescence values in the blue histogram indicate living cells, low values correspond to dead cells. See the following image as an example.
When using the calcein marker in the blue histogram for gating, only the living cells are considered for building the red histogram. High red fluorescence values indicate living, apoptotic cells, low red fluorescence values indicate living, non-apoptotic cells. See the following example.
Dot plot evaluation If you switch to the Dot Plot tab, one region is displayed in the dot plot. The red fluorescence values of the region are related to the marker in the red histogram, the blue fluorescence values to the marker in the blue histogram. As in the histogram evaluation, high blue fluorescence and high red fluorescence represent living cells with annexin V binding. See the following example.
The results of the dot plot evaluation are displayed in the result table.
Evaluating GFP Assays With GFP (Green Fluorescent Protein) assays, the fluorescent substance is not a dye, but a protein. Cells can be transfected with a target gene together with the GFP-producing gene. Transfected cells produce the fluorescent protein, which can be detected. The fluorescence shows you the success of the transfection experiment.
High fluorescence value indicates high amount of CBNF-stained cells Low fluorescence value indicates dead cells The values are displayed in the result table, each histogram has its own table. All events related to the red marker (here CBNF) All measured events After gating by using the red histogram, in the blue histogram only CBNF-stained cells are displayed. High blue fluorescence values indicate GFP-producing cells. See the following example.
High fluorescence value indicates GFP-producing cells Amount of the CBNF containing cells in relation to all measured cells Amount of all cells with high CBNF fluorescence selected by the red marker Amount of cells with high GFP fluorescence in relation to the amount of CBNF stained cells Contents ▲ 242 ▼ Index
Dot plot evaluation If you switch to the Dot Plot tab, one region is displayed in the dot plot. The red fluorescence values of the region are related to the marker in the red histogram, the blue fluorescence values to the marker in the blue histogram. Corresponding to the histogram evaluation, high blue fluorescence and high red fluorescence indicate living GFP-producing cells. See the following example.
The results of the dot plot evaluation are displayed in the result table. Events covered by the region All measured events Amount of cells with high CBNF fluorescence and high GFP fluorescence in relation to all measured events Amount of cells with high GFP fluorescence in relation to the amount of CBNF-stained cells.
Working with Chip and Assay Data You can make efficient use of the chip and assay data generated by the 2100 expert software, if you know the following fundamentals and operating techniques: • “2100 Expert Data Overview” on page 246 • “Handling Assays” on page 249 • “Handling Chip Data” on page 253 • “Organizing, Retrieving, and Backing up 2100 Expert Data” on page 255 • “Importing Data” on page 256 • “Exporting Data” on page 263 • “Printing Reports” on page 275 • “Configuring Tables” on page 282 • “Readin
2100 Expert Data Overview The 2100 expert software manages data in the following different formats: • Assay files (.xsy) • Chip data files (.xad) • Comparison files (.xac) • Validation results files (.xvd) • Diagnostics results files (. xdy) • Result flagging rule files (.xml) Assay files Assay files (.xsy) contain the following information: • Data analysis setpoints Setpoints are instrument commands, data acquisition parameters, and evaluation parameters, some of which you can modify.
• Ladder table and peak table (electrophoretic assays only) • Result flagging rules (electrophoretic assays only) Chip data files Chip data files (.xad) contain the following information: • Measurement results After each chip run, the measurement results—also called “raw data”—are automatically saved in a new chip data file.
Comparison files You can compare the measurement results from different chip runs (electrophoretic chip data files of same assay class only) by collecting samples from different chip data files (.xad) and storing them in a comparison files (.xac). It is then possible to overlay electropherograms of these samples, for example. Validation results files Validation results files (.xvd) contain results of qualification tests regarding the bioanalyzer hardware and software. The files are stored in the “..
Handling Assays Predefined Assays Predefined assays are provided with 2100 expert. They are meant and prepared for measurements using the available LabChip kits. Predefined assays such as Apoptosis or DNA 1000 are write-protected. Although you can open predefined .xsy files and edit some of their properties, you cannot save any changes under the original file name. Custom Assays However, you can derive your own assays from predefined assays as described in “How to Create a Custom Assay” on page 251.
TIP If you just want to view the properties of a custom assay, you can open the assay file in read-only mode (see “Open” on page 514), ensuring you do not make accidental changes. The Assays menu (see “Assays Menu” on page 337) is dynamically built from the structure and contents of the “..\assays” subdirectory of the 2100 expert installation folder. TIP You can add items to the Assays menu by placing assay (.xsy) files—your own assays, for example—in subdirectories of the “..\assays” directory.
How to Create a Custom Assay To create a custom assay: 1 Switch to the Data and Assay context. 2 From the Assays menu, select an assay. – OR – Select File > Open ... and open an assay (.xsy) or chip data (.xad) file. The file appears in the Tree View Panel. NOTE If you want to create a new flow cytometric assay with free gating direction or with more than one marker or region, open and modify the assay “Generic.xsy”.
How to Modify a Custom Assay NOTE You cannot save modifications to predefined assays such as Apoptosis or DNA 1000. To modify a custom assay: 1 From the File menu select Open .... The Open dialog box appears. 2 Select an assay (.xsy) file and click Open. The assay appears in the Tree View Panel and the Assay Properties Tab is displayed. 3 Modify the assay by making changes on the following tabs: – Modify assay setpoints on the Assay Properties Tab.
Handling Chip Data Chip data (.xad) files are automatically generated at the end of a chip run. The .xad files are given names that correspond to the choices you have made in the Options dialog box (see “How to Specify Data File Names and Directories” on page 294). Modifying and saving chip data files 2100 expert allows to re-open chip data files, reanalyze them using different evaluation parameters and store the new results.
The benefit of opening chip data files as read-only is to prohibit you or other users from making changes that would alter the file in any way. Because the 2100 expert software allows you to open chip data files, modify data, and save them, you may prefer to ensure that the original parameters that were used to create the file are not altered.
Organizing, Retrieving, and Backing up 2100 Expert Data As you begin to work with the 2100 expert software, it is good practice to organize your files. If you are not the only user of the bioanalyzer, creating a directory within which to save your files is recommended; having each person save files to their own directory will speed the process of finding a particular file when someone wishes to examine the data again.
Importing Data 2100 expert allows to reprocess assays and chip run files from the Bio Sizing and Cell Fluorescence applications. This is described in: • “Importing Bioanalyzer Files” on page 257 When working with assay (.xsy) or chip data (.xad) files, you will enter specific information that you wish to reuse.
Importing Bioanalyzer Files NOTE Bioanalyzer files imported and saved in the 2100 expert software can no longer be opened with the original programs (Bio Sizing and Cell Fluorescence). However, these bioanalyzer files will not be overwritten, because 2100 expert uses a different file extension. To import Bio Sizing or Cell Fluorescence acquired files: 1 Switch to the Data and Assay context. 2 From the File menu select Import... to display the Open dialog box. 3 Select a file of type .asy, .csy, .cld, or .
Upon importing, the file gets converted to a new 2100 expert file: Bioanalyzer files of these formats are converted to the following: .asy (Bio Sizing assay) .xsy (2100 expert assay) .cld (Bio Sizing chip data) .xad(2100 expert chip data) .csy (Cell Fluorescence assay) .xsy (2100 expert assay) .cad (Cell Fluorescence chip data) .xad (2100 expert chip data) Note that the new 2100 expert file inherits the name from the old bioanalyzer file, but not the extension.
Importing Data Analysis Setpoints You can import data analysis setpoints from other assay (.xsy) or chip data (.xad) files of the same type. Note the following when importing: • Electrophoresis files to be imported must be of the same assay type. This means that you cannot import setpoints from a DNA 1000 assay into a DNA 500 assay, for example. • Flow cytometry files to be imported can be of any flow cytometric assay type, but the import will change the type of the current file to Generic.
4 Click Yes. NOTE Importing data analysis setpoints overwrites all current setpoint values. – All files: the setpoint values are updated in the setpoint explorer (see “Assay Properties Tab” on page 418), and immediately applied to the measurement results (if any).
Importing Chip, Sample, and Study Information On the Sample Information and Study Information sub-tabs of the Chip Summary tab, you can enter names and comments regarding chip, samples, and study. The information you enter here may be very similar for further chip runs or other assays. Once you have entered the information, you can export it into a separate file (see “Exporting Chip Run Data” on page 264), which you can then import into other chip data (.xad) or assay (.xsy) files instead of typing it anew.
Importing Result Flagging Rules You can import result flagging rules into electrophoretic assay (.xsy) or chip data (.xad) files. Result flagging rules are stored in .xml files (see “Exporting Result Flagging Rules” on page 274). To import result flagging rules: 1 Select the Data and Assay context and load an electrophoretic assay or chip data file. 2 Switch to the Result Flagging Tab. 3 In the Result Flagging toolbar click on . The Load Rules dialog box appears. 4 Select the .
Exporting Data 2100 expert allows you to export the results of your chip runs in a variety of formats. The exported data can be used for further evaluation with other applications, such as text processors, graphic tools, MS Excel®, or flow cytometry applications.
Exporting Chip Run Data To export chip run data: 1 Switch to the Data and Assay context. 2 In the Tree View Panel, select a chip data (.xad) .file or load a file. 3 From the File menu, select Export... If you selected an electrophoretic chip data file, the Export Options (Electrophoresis) dialog box appears. If you selected a flow cytometric chip data file, the Export Options (Flow Cytometry) dialog box appears. 4 Select the export categories, and specify a target directory.
TIP Chip run data can be automatically exported every time a chip run has finished. Refer to “Exporting Chip Run Data Automatically” on page 266 for details.
Exporting Chip Run Data Automatically NOTE Keep in mind that exporting a chip data file can require up to 20 MB of disk space. In particular, exporting electropherograms and gel-like images as .tif or .bmp files may take up a lot of disk space. To enable and configure automatic export: 1 From the Tools menu select Options.... The Options dialog box appears. 2 Select the Advanced tab. 3 Select Auto Export and click on the Settings... button next to this option. The Auto Export dialog box appears.
Exporting Tables You can export: • Result tables, peak tables, fragment tables, and ladder tables as .csv files or .xls files. • Log book tables as .html or .txt files. To export a result table, peak table, fragment table, or ladder table: 1 On the Assay Properties, Electropherogram, Gel, Histogram, or Dot Plot tab, right-click the heading row of a table. 2 From the context menu, select Export.... The Save As dialog box appears. 3 Enter a file name and choose the destination directory. 4 Select .csv or .
3 Enter a file name and choose the destination directory. 4 Under Export file format, select HTML file for .html output, or Tabbed text file for .txt output. 5 Click OK.
Exporting Graphs You can export the following graphs as individual graphic files: • Gel-like image • Electropherogram • Electropherogram overview • Histogram • Dot plot • Standard curve • Calibration curve • Raw signals (during chip run) To export a graph: 1 Right-click the graph, and select Copy Graph to File from the context menu. – OR – Click on the button in the toolbar. The Save As dialog box appears. 2 Under File name, enter a name and choose the destination directory.
Note the following: • Histograms: only one histogram graph is exported, either the red or the blue histogram. • Electropherograms: if the grid view is active, an overview image of the electropherograms (of all samples and the ladder) is exported. TIP Electropherograms, gel-like images, histograms, and dot plots can be automatically exported every time a chip run has finished. Refer to “Exporting Chip Run Data Automatically” on page 266 for details.
Putting Graphs and Tables on the Clipboard You can put the following items on the clipboard: • Gel-like image • Electropherogram • Electropherogram overview • Histogram • Dot plot • Standard curve • Calibration curve • Raw signals (during chip run) Copying a graph causes a device-independent bitmap to be placed on the clipboard.
To put a graph or table on the clipboard: 1 Right-click the graph or table (region). 2 From the context menu, select Copy Gel/Copy Electropherogram (graphs) or Copy To Clipboard (tables). – OR – Click the button in the toolbar. You can now switch to a word processing, spreadsheet, graphics, or other application, and paste the graph or table there.
Exporting Chip, Sample, and Study Information On the Sample Information and Study Information sub-tabs of the Chip Summary tab, you can enter names and comments regarding chip, samples, and study. The information you enter here may be very similar for further chip runs or other assays. Once you have entered the information. you can export it into a separate file, which you can then import into other chip data (.xad) or assay (.xsy) files instead of typing it anew.
Exporting Result Flagging Rules You can export result flagging rules for reuse in other electrophoretic assay (.xsy) or chip data (.xad) files (see “Importing Result Flagging Rules” on page 262). Result flagging rules are stored in .xml files. To export result flagging rules: 1 Select the Data and Assay context and load an assay or chip data file. – OR – In the Tree View Panel, select the electrophoretic assay or chip data file that contains the result flagging rules. 2 Select the Result Flagging Tab.
Printing Reports For documentation and presentation purposes, you can print reports for assay (.xsy), chip data (.xad), validation results (.xvd), and comparison (.xac) files. You can print all reports manually, see “How to Print a Chip Run Report” on page 276. When printing manually, a preview function allows you to view the printout before starting the print job. The 2100 expert program can also be set to print customized chip run reports automatically at the end of the run.
How to Print a Chip Run Report The following information can be included in a chip run report: • You can always include: – Assay summary—general data about the assay, and sample information. – Assay details—complete list of data analysis setpoints. • For chip data files (.xad) you can include: – Chip run summary—general information on the chip run. • For flow cytometric chip data files (.xad) you can include: – Histogram summary—all histograms shown in an overview.
To print a report: 1 Switch to the Data and Assay context. 2 In the Tree View Panel select a file, either an assay (.xsy) or a chip data (.xad) file. 3 From the File menu select Print.... Depending on the file type different dialog boxes appear. 4 Under Print Item and Samples, select what you want to print. You can print just one or any combination of the items shown in this section by enabling the desired options.
wells you would like to print, following the instructions as shown on the dialog box. Also refer to “Print (Electrophoresis)” on page 531/“Print (Flow Cytometry)” on page 528 for details. NOTE Your selections here are separate from the Auto Print selections (they do not affect each other). Both are used by default the next time you print (even after restarting the program). 5 Use the Page Setup... and Printer...
The following example shows the “Run Summary” part of an RNA chip run report.
How to Turn on and Configure Automatic Printing of Chip Run Reports A report can be printed (or generated) automatically at the end of each chip run. To enable and configure automatic printing: 1 From the Tools menu select Options.... The Options dialog box appears. 2 Click the Advanced tab to bring it to the front. 3 Select the Auto Print check box and click the Settings... button next to this check box. The Auto Print dialog box appears.
4 Adjust the settings: – Under Print Item, select the options that should be contained in the report. – Under Save To File, you can redirect the automatic printouts to .pdf and .html files. Note that no print output is generated if you select the PDF and/or HTML option. – Using the Page Setup... and Printer... buttons, you can access system dialog boxes, allowing you to select a printer for the automatic print, and specify the print medium and page layout.
Configuring Tables 2100 expert uses various tables to present data: • Result tables • Peak tables • Fragment tables • Log book tables In some cases, you might want to reorganize the way the data is presented. To do so, you can hide or show columns, change the column sequence, and adapt the table height. The following example demonstrates how to add the migration time to the Peak Table.
Showing and Hiding Columns To add the Aligned Migration Time column to the table: 1 Right-click the heading row of the table. 2 From the context menu, select Configure Columns... to display the Configure Columns dialog box.
3 Move Aligned Migration Time from the Available list to the Displayed list by clicking the single-arrow button. 4 Click OK. A new column Aligned Migration Time is inserted in the table: To hide columns, remove their names from the Displayed list in the Configure Columns dialog box.
Changing the Column Sequence TIP You can set the column sequence also using the Up and Down buttons in the Configure Columns dialog box. To change the column sequence of a table: 1 Position the mouse pointer on a column header. 2 Click and hold the left mouse button, and drag the header cell to the desired position. While dragging, a green arrow indicates the target position. 3 Release the mouse button.
Changing the Table Height To increase or reduce the table height: 1 Position the mouse pointer above the heading row of the table until the cursor’s shape changes to a double arrow. 2 Click and hold the left mouse button and drag up or down. 3 Release the mouse button.
Reading the Log Books 2100 expert provides two log books: Run Log The run log table contains events generated during a chip run, including the start and end time and any errors or problems that occurred during the run. You can view the run log table in the Data and Assay context by selecting the Log Book tab. Please refer to “Log Book Tab” on page 481 for more details on the run log table. The run log is saved in the chip data (.xad) file and cannot be cleared.
Please refer to “System Log Viewer” on page 561 for more details on the system log table. The system log is saved in the file “SystemLogBook.log” located in the “..\log” subdirectory. The system log file can grow very large and fill up a lot of worthy disk space. To control the disk space occupied by the system log file, read “How to Use the Advanced Settings” on page 299.
How to Change the Display of the Log Books To sort a log book table: 1 Right-click any table column. 2 From the context menu, select Sort to sort the table by the current column (ascending). – OR – From the context menu, select Sort by Event to sort the table by the Category column (ascending). To filter a log book table: 1 In the Log Book toolbar, click on Filter . The Filter Events dialog box appears.
The filter definition in the following example would exclude all events from the log book table with an Event Type other than Critical. For more information refer to “Log Book Tab” on page 481. 4 Click OK to apply the filter to the log book table. To remove the filter from a log book table: 1 In the Log Book toolbar, click on Reset Contents .
TIP You can hide/show any of the log table columns, and re-sort the columns by right-clicking the table and selecting Columns... from the context menu. See “Configuring Tables” on page 282. How to Search the Log Book You can search both the Run Log and the System Log for any string. Using the example of the Run Log this is demonstrated below. To search the Log Book: 1 In the Log Book toolbar, click on Find . The Find dialog box appears. 2 Enter a search string and select the search Direction.
NOTE The search is not case-sensitive. 4 To continue the search, click Find Next.
Configuring 2100 Expert The 2100 expert software can be configured as follows. • The names of data files can be generated automatically. Learn how to determine the naming mechanism in “How to Specify Data File Names and Directories” on page 294. • When a chip run is complete, you have to remove the chip from the bioanalyzer. You can activate an acoustic alert that reminds you to do that. “How to Set the Acoustic Chip Alert” on page 296 gives you the details.
How to Specify Data File Names and Directories The measurement results are stored automatically when the chip run is complete. To make it easier for you to identify the chip data files, you can configure an automatic naming scheme for the files. To specify the names and destination for generated chip data files: 1 Select Tools > Options.... The Options dialog box appears with the Data Files tab in front.
2 Select the check boxes of the strings you want to insert in the file names: Option Meaning Prefix Inserts an annotation to identify the data file. This string can be modified. The default file prefix is “2100 expert”. Assay Class Inserts the assay class in the file name. Examples: “DNA1000”, “GFP”, “Apoptosis”. Serial Number Inserts the serial number of the Agilent 2100 bioanalyzer instrument used for the chip run. Date Inserts the date of the chip run.
How to Set the Acoustic Chip Alert When a chip run has finished, you have to remove the chip from the bioanalyzer. You can activate an acoustic alert sound that reminds you to do this. To set the chip alert sound: 1 Select Tools > Options... to display the Options dialog box. 2 Click the Chip Alert tab to bring it to the front.
3 Select Off to turn off the alert sound. – OR – Select Default sound to use the standard alert sound. – OR – Select Custom Sound to specify a .wav file to be used as alert sound. 4 Click Play to test the alert sound. 5 Use the Alert interval slider to specify the intervals the alert should be triggered. The interval can be set within the range of 1 to 15 seconds. 6 Click OK to confirm your modifications.
How to Specify Graph Settings You can compare measurement results of samples by overlaying their electropherograms or histograms. For optimal display, you can configure the curve colors and adjust the scale in overlaid graphs. To modify the graph settings: 1 Select Tools > Options... to display the Options dialog box. 2 Click the Graph Settings tab to bring it to the front. 3 Click the colored rectangles to the right of the signals. You can now choose a new color in the Color dialog box.
How to Use the Advanced Settings You can use 2100 expert’s advanced settings to: • Automate starting, documenting, and archiving of chip runs. Activating the Auto Run, Auto Print, Auto Export functions is especially useful when running a series of the same assay. • Set disk space limits for data storage. Disk space limitation for raw data and log files protects you from data overflow when performing large numbers of chip runs. • Specify correction rules for electrophoretic measurement data.
To modify the advanced settings: 1 Select Tools > Options... to display the Options dialog box. 2 Click the Advanced tab to bring it to the front. 3 Select Limit the storage of raw data backups ... and enter an upper limit (in MB) if you want to limit disk space for the storage of packet files. 20 MB (~ 20 chip runs) is the default.
NOTE In addition to the regular chip data file (.xad), 2100 expert creates another raw data backup file (packet file: .pck) with each chip run. The size of a typical packet file is about 1 MB. It is stored in the “..\data\packets” folder of the installation directory. You can set the limit for the disk space to be used for packet files. In case this limit is reached, the oldest file(s) will be deleted to get free disk space (first in, first out).
NOTE The Auto Print and Auto Export settings are independent from those made via the Export or Print command of the File menu. 8 Click OK to confirm your modifications.
Running Instrument Diagnostics 2100 expert provides several tests to check proper functioning of the bioanalyzer hardware. You should perform the tests on a regular basis, or if incorrect measurements occur.
Diagnostics Test Purpose Fan Test Checks if the fan is running at the appropriate speed. Temperature Test Checks if the temperature ramp-up speed of the heater plate is within specifications. Electrode Cartridge Tests Diagnostics Test Purpose Current Leakage Test Measures electrode cartridge leak current(s) between pins. Leak current test chip required. For correct preparation of the leak current test chip, please refer to the technical note included in the test chip bundle.
Diagnostics Test Purpose High-Voltage Accuracy Check of channel-reference diode in transmission direction. on Load Test Autofocus Test Checks the focusing capability of the optical system. Autofocus test chip required. Laser Stability Test Measurement of stability of red laser signal. Electrode/Diode Test Checks the photo diode and current-versus-voltage performance of the bioanalyzer. Electrode/diode test chip required.
Diagnostics Test Purpose System Leak Test Checks if the bioanalyzer is able to maintain a vacuum. Produces a test pressure of -100 mbar and monitors for changes. Cell Autofocus test chip required. Cell Autofocus Test Checks that the optical system of the bioanalyzer is correctly calibrated. Cell Autofocus test chip required. NOTE With bioanalyzer model G2938A only diagnostic tests in combination with the electrode cartridge can be performed.
Test Chip Comment Electrode/Diode Test Chip Can be used multiple times. Leak Current Test Chip Quantity 1 Has to be prepared with deionized water. Can be 5 only used once. Test Chip Kit for Flow Cytometry Assays (reorder no. G2938-68200) Test Chip Comment Quantity Cell Autofocus Test Chip Required for Pressure Control Test, System Leakage Test, and Optical Drive Test. Can be used multiple times.
How to Run Instrument Diagnostics Tests NOTE Diagnostics tests cannot be run while the 2100 expert software is performing a chip run. To run diagnostics tests: 1 From the Context menu, select Instrument. 2 In the Tree View Panel, select the bioanalyzer on which you want to run the tests. 3 Select the Diagnostics tab. All available tests are displayed in the Available Tests list.
4 Select the tests you want to run: – Select the Apply check boxes to select single tests. – Click Select All to select all available tests. – Click Unselect All to deselect all tests. 5 Click Start.
6 Follow the instructions given by the 2100 expert software. For example, exchange the cartridge, or put a test chip in the receptacle of the bioanalyzer when requested by the software. All selected tests are performed.
The Status column indicates the status of each test: – Executing – Execution pending – Executed, passed – Executed, failed 7 If any test failed, redo the test. 8 If failures still persist, contact Agilent service. The results of diagnostics tests are stored in .xdy files in the 2100 expert installation folder under “..\diagnosis”. If tests fail, send the .xdy files to the Agilent service.
Performing Qualifications To ensure a validated Agilent 2100 bioanalyzer system, qualification steps have to be performed at installation and operation level. 2100 expert allows for detailed installation qualification (IQ) and operational qualification (OQ) on both the bioanalyzer hardware and software. Each qualification comprises a series of tests and measurements that you can run and document in the Validation context of the 2100 expert software.
Operational Qualification Operational qualification proves that the bioanalyzer system is suitable for its intended use, that is, that it will function according to its operational specifications in the selected environment. Operational qualification should be performed: • at first use of the instrument, • after relocating the instrument, • after changing essential parts of the system, for example software updates or exchange of cartridges, • after instrument repair, • on regular time intervals.
Validation Procedure To perform qualification tests: 1 Switch to the Validation context. 2 From the File menu select New. 3 A New Validation item appears in the Tree View Panel. 4 Under Cartridge Details, click on Select... and specify details on the cartridge that is currently installed in the bioanalyzer.
6 In the Tree View Panel, navigate to the test category you want to execute. Select the category via IQ/OQ – SW/HW – PC name/Bioanalyzer name – Test Category. NOTE To execute hardware tests (HW branch) the bioanalyzer must be properly connected and switched on. The Configuration tab now lets you select qualification tests to be executed in the validation run: To select tests, check the Apply check box next to the test(s).
The Save As dialog box appears. 8 Specify a name and location for the validation results file (.xvd) and click Save. The selected tests are executed.
10Click OK. 11The Status column shows whether the tests have been run successfully or not. For details on the Configuration tab, please refer to “Configuration Tab” on page 485.
12To view details on test execution, select the Results tab. For details on the Results tab, please refer to “Results Tab” on page 487. 13You can now navigate to other test categories and execute additional qualification tests.
15When you close the validation results file (File > Close), try to switch to another context or exit 2100 expert, the following message appears: If you select No, you return to the validation context and can run further qualification tests. If you select Yes, the validation results file (.xvd) is closed and becomes read-only. NOTE You can re-open validation results files only for viewing and printing. TIP Select File > Print... to generate a printed report of the validation run.
2100 Expert Software Reference The 2100 expert software reference is organized as follows: • 2100 Expert Application Window Elements, such as menus, toolbars, status bar, panels, and tabs establish a graphical standard user interface. For details, refer to “2100 Expert Application Window Elements” on page 321. • Dialog Boxes are used for various file operations, for controlling and analyzing chip runs, and for configuring the 2100 expert application. For details, refer to “Dialog Boxes” on page 494.
2100 Expert Application Window Elements The 2100 expert application window has the following regions (Data and Assay context): Contents ▲ 321 ▼ Index
Region Purpose Title Bar Bar at the top of the window providing information on the application that you have started, and the assay or chip file you have selected. Menu Bar Bar beneath the title bar that contains pull-down menus. Toolbars Provides quick access to the most often needed commands.
Region Purpose Lower Panel Displays a chip icon/a small gel view that you can use to navigate through your samples. Status Bar Shows system messages and activities, the current status of the application, and whether or not Auto Run, Auto Export and Auto Print are activated. Title Bar If you are in the Data and Assay context, name and path of the current chip data or assay file are displayed on the title bar: If the file has been opened as read-only, this is shown in parentheses.
Menu Purpose Assays Menu Lets you open assay files for editing (Data and Assay context). Lets you select an assay for a chip run (Instrument context). Gel Menu Provides functions for changing the display of gel graphs. Electropherogram Provides functions for changing the display of electropherograms, Menu and for overlaying electropherograms. Histogram Menu Provides functions for changing the display of histograms, overlaying histograms, and for handling markers and gates.
Menu Purpose Windows Menu Lets you switch between open chip data or assay files (Data and Assay context). Lets you select one of the connected bioanalyzers (Instrument context). Help Menu Lets you access Help, get information about the installed software and hardware, and register additional software options. NOTE Not all menus are visible at the same time. The Electropherogram menu, for example, is only available if electropherograms (single or grid view) are displayed.
File Menu The File menu provides functions for file handling and printing.
File menu item Function Open... Brings up the Open dialog box, allowing you to open a chip data (.xad) or assay (.xsy) file. Multiple files of different assay types can be opened in parallel. Refer to “Open” on page 514. Close Closes the currently selected chip data or assay file. Close All Closes all open chip data and assay file files. Save Saves the selected chip data or assay file under its current name. Save As...
File menu item Function Print... Displays the Print (Flow Cytometry) or Print (Electrophoresis) dialog box, allowing you to generate various printouts of the selected chip data or assay file. Refer to “Print (Flow Cytometry)” on page 528 and“Print (Electrophoresis)” on page 531. Recently used files A list of up to ten chip data (.xad) and/or assay (.xsy) files gives you quick access to the most recently used files. Exit Exits the 2100 expert application.
Instrument Context File menu item Function Open File to Run... Brings up the Open dialog box, allowing you to select a chip data (.xad) or assay (.xsy) file for a chip run. Refer to “Open” on page 514. Recently used files A list of up to ten chip data (.xad) and/or assay (.xsy) files gives you quick access to the most recently used files. Exit Exits the 2100 expert application. If there is unsaved data, the 2100 Expert – Close dialog box appears. Refer to “2100 Expert – Close” on page 499.
Validation Context File menu item Function New Begins a new validation and inserts a New Validation item in the tree view. Open... Brings up a system dialog box, allowing you to open a validation results (.xvd) file. Close Closes the current validation results file. Close All Closes all open validation results files. Page Setup... Displays the Page Setup dialog box, allowing you to change the layout of the printed page(s). Refer to “Page Setup” on page 555.
File menu item Function Print... Displays the Print (Validation) dialog box, allowing you to generate various reports on qualification tests. Refer to “Print (Validation)” on page 535. Recently used files A list of up to ten validation results (.xvd) files gives you quick access to the most recently used files. Exit Exits the 2100 expert application. If there is unsaved data, the 2100 Expert – Close dialog box appears. Refer to “2100 Expert – Close” on page 499.
File menu item Function Open... Brings up a system dialog box allowing you to open a comparison (.xac) or chip data (.xad) file. Close Closes the current comparison file. Close All Closes all open comparison files. Save Saves the selected comparison file under its current name. Save As... Opens a system dialog box allowing you to save the current comparison file under a new name. Page Setup... Displays the Page Setup dialog box, allowing you to change the layout of the printed page(s).
Context Menu The Context menu lets you switch between the four program modes, called “contexts”: Context menu item Function Instrument Switches to the Instrument context. Data and Assay Switches to the Data and Assay context. Validation Switches to the Validation context. Comparison Switches to the Comparison context. The checkmark indicates the active context.
View Menu The View menu provides functions to switch between the Gel/Electropherogram and Histogram/Dot Plot views, and between single- and multi-instrument view. It also lets you show and hide panels. Data and Assay context (electrophoretic assay selected) View menu item Function Gel Switches to the Gel Tab of the current chip data (or assay) file. Electropherogram Switches to the Electropherogram Tab (Single/Grid View) of the current chip data (or assay) file.
Data and Assay context (flow cytometric assay selected) View menu item Function Histogram Switches to the Histogram Tab (Single/Grid View) of the current chip data (or assay) file. Dotplot Switches to the Dot Plot Tab (Single/Grid View) of the current chip data (or assay) file. Context Bar Shows or hides the Context Bar. Tree View Shows or hides the Tree View Panel. Lower Panel Shows or hides the Lower Panel (chip graphic or gel graphic).
Instrument context View menu item Function Single View Switches to the Instrument Tab (Single View) of the selected instrument. Grid View Switches to the Instrument Tab (Grid View). Context Bar Shows or hides the Context Bar. Tree View Shows or hides the Tree View Panel. Validation/Comparison context View menu item Function Context Bar Shows or hides the Context Bar. Tree View Shows or hides the Tree View Panel.
Assays Menu From the Assays menu, you can open assay files for editing (Data and Assay context). In the Instrument context, you can select an assay for a chip run if a bioanalyzer is connected and switched on. Assays menu item Function Electrophoresis – dsDNA All assays located in the ”..\assays\electrophoresis\dsDNA” folder are listed here.
Assays menu item Function Electrophoresis – RNA All assays located in the ”..\assays\electrophoresis\RNA” folder are listed here. By default, these are: • Eukaryote Total RNA Nano • Eukaryote Total RNA Pico • mRNA Nano • mRNA Pico • Prokaryote Total RNA Nano • Prokaryote Total RNA Pico Electrophoresis – Protein All assays located in the ”..\assays\electrophoresis\protein” folder are listed here.
Assays menu item Function Flow Cytometry All assays located in the ”..\assays\flow cytometry” folder are listed here.
Assays menu item Function Demo – Flow Cytometry All assays located in the ”..\assays\demo\flow cytometry” folder are listed here. By default, these are: • Antibody Staining (Demo) • Apoptosis (Demo) • Checkout Beads (Demo) • GFP (Demo) • Generic (Demo) You can use these assays for training in offline mode (with no bioanalyzer connected). See “Running a Demo Assay” on page 41.
Assays menu item Function Demo – Electrophoresis All assays located in the ”..\assays\demo\electrophoresis” folder are listed here. Demo Cy5 Labeled Nucleic Acids Nano • Demo DNA 1000 • Demo DNA 12000 • Demo DNA 500 • Demo DNA 7500 • Demo Eukaryote Total RNA Nano • Demo Eukaryote Total RNA Pico • Demo mRNA Nano • Demo mRNA Pico • Demo Prokaryote Total RNA Nano • Demo Prokaryote Total RNA Pico • Demo Protein 200 Plus • Demo Protein 50 Use these demos for offline training (with no bioanalyzer connected).
Gel Menu Gel menu item Function Automatic Analysis Starts automatic analysis. Peaks are detected, and the Peak Table gets calculated. Results are getting recalculated whenever changes to the data analysis setpoints are applied. Pause Automatic Pauses automatic analysis. It is recommended to pause analysis Analysis during manual integration.
Gel menu item Function Don’t Analyze Analysis is switched off. Raw data is displayed on the Gel tab and the Peak Table is cleared. Previous Sample Highlights and displays the gel graph of the previous sample. If the ladder is highlighted, jumps to the last sample. Not available if the first sample is highlighted. Next Sample Highlights and displays the gel graph of the next sample. If the last sample is highlighted, jumps to the ladder. Not available if the ladder is highlighted.
Gel menu item Function Gel Color Opens a submenu from which you can select a predefined foreground/background color scheme to be used for displaying the gel graph (all lanes). The colors are designed to approximate various actual gel staining and imaging techniques. Blue on White, for example, simulates a Coomassie gel often used with proteins.
Electropherogram Menu Contents ▲ 345 ▼ Index
Electropherogram Function menu item Automatic Analysis Starts automatic analysis. Peaks are detected, and the Peak Table gets calculated. Results are getting recalculated whenever changes to the data analysis setpoints are applied. Pause Automatic Analysis Pauses automatic analysis. Don’t Analyze Analysis is switched off. Raw data is displayed on the Electropherogram tab and the Peak Table is cleared. Previous Sample Highlights and displays the electropherogram of the previous sample.
Electropherogram Function menu item Scaling Mode Opens a submenu from which you can select one of the following modes: • Individual Scale Each graph uses its own scaling which is optimized for that electropherogram. • Selected Scale All graphs use the same scaling which is optimized for the selected graph. • Global Scale (only available in grid view). All graphs use the same scaling which is chosen such that all electropherograms fit into the display.
Electropherogram Function menu item Graph Mode Lets you select one of the following click-and-drag operations on electropherograms: • Zoom Magnifies the corresponding region to the full display. • Pan Moves the graph, makes especially sense for already zoomed graphs. • Scale Dragging left or down shrinks the display in that direction, dragging right or up magnifies the display in that direction.
Electropherogram Function menu item Copy Electropherogram Puts a copy of the electropherogram into the clipboard (single-well view). In multi-well view, all electropherograms are copied. Save Electropherogram Opens a system dialog box, allowing you to save the electropherogram (single-well view) or all electropherograms (multi-well view) as an image in JPEG (.jpg), Windows Bitmap (.bmp), Windows Meta File (.wmf), CompuServe Graphics Interchange (.gif), or Tagged Image File (.tif) format.
Electropherogram Function menu item Overlaid Samples Lets you overlay electropherograms from multiple wells (single-well view only). Each electropherogram will be shown in a different color and a color legend appears on the Legend sub-tab. • Sample 1...12 selects an individual sample to be overlaid with the current sample (which is grayed out). • Ladder overlays the electropherogram of the ladder well. • No Overlay undoes overlaying. • All Samples overlays all samples of the chip.
Histogram Menu Single View Contents ▲ 351 ▼ Index
Histogram menu item Function View Single Sample Displays the current histogram in single view. View All Samples Switches to the grid view and highlights the current histogram. Undo Zoom Undoes the last zoom action. Undo All Undoes all zoom actions. Scale to all signals Adapts the scales of the X and the Y axis to all overlaid graphs. All curves will be completely visible in overlaid graphs. Gradient Puts a gray-to-white gradient on the background of the histogram(s), or removes the gradient.
Histogram menu item Function Overlaid Samples Lets you overlay histograms from multiple samples (single view only). Each histogram will be shown in a different color and a color legend appears above the graph. • Sample 1...6 selects an individual sample to be overlaid with the current sample (which itself is not available in the menu). • No Overlay undoes overlaying. • All Samples overlays all samples of the chip. Insert Marker Inserts a marker into the histogram.
Histogram menu item Function Gate in Red histogram Uses the selected marker of the blue histogram for gating in the red histogram (generic assays only). Insert Gate into All Uses the selected marker of the blue histogram for gating in all red Red histograms histograms (generic assays only). Gate in blue histogram Uses the selected marker of the red histogram for gating in the blue histogram (generic assays only).
Grid View In grid view, the Histogram menu has the following commands, which work the same way as in single view (see above): Contents ▲ 355 ▼ Index
Dot Plot Menu Single View Contents ▲ 356 ▼ Index
Dot Plot menu item Function Single View Displays the current dot plot in single view. Grid View Switches to the grid view and highlights the current dot plot. Undo Zoom Undoes the last zoom action. Undo All Undoes all zoom actions. Gradient Puts a gray-to-white gradient on the background of the selected dot plot, or removes the gradient. Copy Graph To Clipboard Puts a copy of the selected dot plot into the clipboard.
Dot Plot menu item Function Insert Region into all dot plots Copies the selected region into all other dot plots of the chip run (generic assays only). Insert Existing Region Opens the Insert Existing Region dialog box (see “Insert Existing Region” on page 551), which allows you to insert existing regions from other dot plots of the chip run in the current dot plot (generic assays only). Horizontal Gate Inserts a horizontal gate for the selected region (generic assays only).
Grid View In grid view, the Dot Plot menu has the following commands, which work the same way as in single view (see above): Result Flagging Menu (Electrophoretic Assays only) Contents ▲ 359 ▼ Index
Result flagging menu item Function Load Rules Opens a system dialog box allowing you to load a set of result flagging rules stored in an .xml) file. Save Rules Opens a system dialog box allowing you to save the result flagging rules in an .xml) file. New Rule Adds a new rule. Delete Rule Deletes the selected rule. Move Up Moves the selected rule up one row. Move Down Moves the selected rule down one row. Copy Rule Inserts a copy of the selected rule.
Log Book menu item Function Find Opens the Find dialog box allowing you to search the run log table for any string. Filter Opens the Filter Events dialog box allowing you to hide run log table entries (rows) matching filter criteria you can specify. Reset Filter Removes any filter you applied to the run log table. Instrument Menu Instrument menu item Function Start Starts a chip run.
Tile Menu The Tile menu is available if the Instrument Tab (Grid View) is displayed: Tile menu item Function Tile Horizontal Arranges the instrument panes horizontally. Tile Vertical Arranges the instrument panes vertically. Previous Highlights the previous instrument pane. Next Highlights the next instrument pane.
Tools Menu Tools menu item Function System Log Opens the System Log Viewer dialog box showing system-wide events in the system log table. Options... Opens the Options dialog box allowing you to configure the 2100 expert software.
Windows Menu In the Data and Assay context, the Windows menu lets you switch between all open chip data (.xad) and assay (.xsy) files: In the Instrument context, the Windows menu lets you switch between the instrument grid view (All Instruments) and all detected instruments: If an assay is already selected for an instrument, the assay name appears instead of the instrument name. In the Validation context, the Windows menu lets you switch between all open validation results files (.
If an assay is already selected for an instrument, the assay name appears instead of the instrument name. Help Menu Help menu item Function Contents and Index Opens the home page of the Agilent 2100 Bioanalyzer Help Desk. Bioanalyzer Online Takes you to the Agilent Lab-on-a-Chip Products web pages (Internet connection required). Bioanalyzer User Forum Takes you to the Life Sciences/Chemical Analysis User Forum (Internet connection and login data required).
Help menu item Function Online Store Takes you to the Life Sciences/Chemical Analysis Online Store (Internet connection required). About 2100 expert... Displays the About 2100 Expert dialog box, which shows information about the system and the software version. Refer to “About 2100 Expert” on page 497. Registration... Opens the License Administration Tool dialog box, allowing you to register the software components which you have licensed.
Toolbars The toolbars are located below the menu bar and give you quick access to often required functions. TIP Move the mouse cursor onto a toolbar button and wait a moment. This will display a tool tip that briefly describes its function. If a toolbar button is dimmed then its function is not available at the moment. The toolbars strongly vary depending on the context and on what you have currently selected.
Instrument Context Toolbar Button Function Select an item from this list to switch to another context. Brings up the Open dialog box, which allows you to load a chip data (.xad) or assay (.xsy) file for your chip run. Shows or hides the Tree View Panel. Displays the Instrument Tab (Single View) of the selected instrument. Displays the Instrument Tab (Grid View). Arranges the instrument panes horizontally. Arranges the instrument panes vertically. Highlights the previous instrument pane.
Data and Assay Context – Electrophoresis Toolbar Button Function Select an item from this list to switch to another context. Brings up the Open dialog box, which allows you to load chip data and assay files. Saves the current chip data or assay file. Opens the Print dialog box, allowing you to send chip and assay data to the printer. See “Print (Electrophoresis)” on page 531 and “Print (Flow Cytometry)” on page 528. Shows or hides the Tree View Panel.
Gel View Starts automatic analysis. Peaks are detected, and the Peak Table gets calculated. Results are getting recalculated whenever changes to the data analysis setpoints are applied. Pauses automatic analysis. It is recommended to pause analysis during manual integration. Analysis is switched off. Raw data is displayed on the Gel tab and the Peak Table is cleared. Highlights and displays the gel graph of the previous sample. If the ladder is highlighted, jumps to the last sample.
Opens a system dialog box allowing you to save the gel graph (all samples) as an image in JPEG (.jpg), Windows Bitmap (.bmp) or Tagged Image File Format (.tif) format. Opens a submenu from which you can select a predefined foreground/background color scheme to be used for displaying the gel graph (all lanes). The colors are designed to approximate various actual gel staining and imaging techniques. Blue on White, for example, simulates a Coomassie gel often used with proteins.
Starts automatic analysis. Peaks are detected, and the Peak Table gets calculated. Results are getting recalculated whenever changes to the data analysis setpoints are applied. Pauses automatic analysis. It is recommended to pause analysis during manual integration. Analysis is switched off. Raw data is displayed on the Electropherogram tab and the Peak Table is cleared. Highlights and displays the electropherogram of the previous sample. If the ladder is highlighted, jumps to the last sample.
Lets you select one of the following scaling modes: • Individual Scale Each graph uses its own scaling which is optimized for that electropherogram. • Selected Scale All graphs use the same scaling which is optimized for the selected graph. • Global Scale (only available in grid view). All graphs use the same scaling which is chosen such that all electropherograms fit into the display.
Shows/hides the data points used to generate the electropherogram. Data points are visible only in the single-well view. Data points are 0.05 seconds apart as the time resolution of the data points is 0.05 seconds, which corresponds to their data acquisition rate (of 20 Hz). Shows/hides grid lines (single-well view only). Puts a copy of the electropherogram into the clipboard (single-well view). In multi-well view, all electropherograms are copied.
Lets you overlay electropherograms from multiple wells (single-well view only). Each electropherogram will be shown in a different color and a color legend appears on the Legend sub-tab. • Sample 1...12 selects an individual sample to be overlaid with the current sample (which is gray out). • Ladder overlays the electropherogram of the ladder well. • No Overlay undoes overlaying. • All Samples overlays all samples of the chip.
Data and Assay Context – Flow Cytometry Toolbar Button Function Select an item from this list to switch to another context. Brings up the Open dialog box which allows you to load chip data and assay files. Saves the current chip data or assay file. Opens the Print dialog box, allowing you to send chip and assay data to the printer. See “Print (Electrophoresis)” on page 531 and “Print (Flow Cytometry)” on page 528. Shows or hides the Tree View Panel.
Histogram View Displays the current histogram in single view. Switches to the grid view and highlights the current histogram. Undoes the last zoom action. Undoes all zoom actions. Adapts the scales of the X and the Y axis to all overlaid histograms. All curves will be completely visible in overlaid histograms. Puts a copy of the selected histogram (blue or red) into the clipboard. Opens a system dialog box allowing you to save the selected histogram (blue or red) as an image in Windows Meta File (.
Lets you overlay histograms from multiple samples (single view only). Each histogram will be shown in a different color and a color legend appears above the graph. • Sample 1...6 selects an individual sample to be overlaid with the current sample (which itself is not available in the menu). • No Overlay undoes overlaying. • All Samples overlays all samples of the chip. Inserts a marker into the histogram. Copies the selected marker in all other histograms of the chip run (generic assays only).
Uses the selected marker of the blue histogram for gating in all red histograms (generic assays only). Uses the selected marker of the red histogram for gating in the blue histogram (generic assays only). Uses the selected marker of the red histogram for gating in all blue histograms (generic assays only). Removes the gate (only from the current histogram). Highlights and displays the histogram of the previous sample. Not available if the first sample is selected.
Puts a copy of the selected dot plot into the clipboard. Opens a system dialog box allowing you to save the selected dot plot as an image in Windows Meta File (.wmf), Windows Bitmap (.bmp), or JPEG (.jpg) format. Enters the region drawing mode allowing you to draw a new region in the dot plot. The mouse cursor changes its shape to a crosshair. Deletes the selected region.
Colors the dots inside the selected region using the color of the region border. Highlights and displays the dot plot of the previous sample. Not available if the first sample is selected. Highlights and displays the dot plot of the next sample. Not available if the last sample is selected. Data and Assay Context – Result Flagging Toolbar Button Function Opens a system dialog box allowing you to load a set of result flagging rules stored in an .xml) file.
Button Function Inserts a copy of the selected rule. Validates the result flagging rules and applies them to the chip data (if any). Switches between Normal and Target Mode. Data and Assay Context – Log Book Toolbar Button Function Opens the Find dialog box allowing you to search the run log table for any string. Opens the Filter Events dialog box allowing you to hide run log table entries (rows) matching filter criteria you can specify. Removes any filter you applied to the run log table.
Validation Context Toolbar Button Function Select an item from this list to switch to another context. Begins a new validation and inserts a New Validation item in the tree view. Brings up a system dialog box, which allows you to open a validation results (.xvd) file. Displays the Print (Validation) dialog box, allowing you to generate various reports on qualification tests. Refer to “Print (Validation)” on page 535. Shows or hides the Tree View Panel. Starts a validation run.
Comparison Context Toolbar Button Function Select an item from this list to switch to another context. Brings up a system dialog box allowing you to open a comparison (.xac) or chip data (.xad) file. Saves the current comparison file. Displays the Print (Comparison) dialog box, allowing you to print a comparison report. Refer to “Print (Comparison)” on page 538. Shows or hides the Tree View Panel.
Information Bar The information bar is located below the toolbar. Instrument context If no chip run is in progress, the information bar shows the name of the active bioanalyzer (or “Demo” if the Demo COM port is selected) followed by the selected assay class: During a chip run, the information bar additionally shows the message “Running” and a chip symbol with a flashing number (number of sample currently being measured): After a chip run, you see messages such as “Run aborted by user”, or “Run complete”.
Data and Assay context If the Gel, Electropherogram, Histogram or Dot Plot tab is selected, you see the tab name followed by the sample name (in single view) or “All Samples” (in grid view).
If you stopped a validation run, the information bar looks as follows: If you re-open a validation results file for review, no modifications are possible. This is indicated on the information bar.
Context Bar The context bar is located on the left-hand side of the application window.
Tree View Panel The tree view panel is located on the left-hand side of the application window. TIP To get more screen space in the work area, you can hide the tree view panel by selecting View > Tree View. Instrument Context In the Instrument context, the tree view shows as many instruments as are detected. If an instrument is detected, its serial number is shown: If a chip is detected in the instrument, a chip icon appears in front of the item, identifying the chip type.
When a chip run has finished, a green checkmark appears: If the run was aborted or an error occurred, a red cross appears instead: • Clicking on All Instruments takes you to the Instrument Tab (Grid View). • Clicking on an instrument item displays the Instrument Tab (Single View).
Data and Assay Context In the Data and Assay context you can use the tree view to navigate through all open chip data (.xad) and assay (.xsy) files. NOTE .xad files that have been opened in the Comparison context also appear in the tree view. The colors of the chip icons next to the file names identify the assay type: blue = DNA, green = RNA, violet = protein, orange = flow cytometry.
• Clicking a plus or minus sign next to a chip icon expands or collapses the sample list. • Clicking a chip data or assay item displays the Chip Summary Tab. • Clicking an All Samples item displays the grid view showing gel views, electropherograms, histograms, or dot plots of all samples. • Clicking a sample item (or the ladder) displays the gel view, the electropherogram (single view), histogram (single view), or dot plot (single view) of this sample.
Validation Context In the Validation context you can use the tree view to: • Navigate through validation results files. Clicking a plus or minus sign next to a chip icon expands or collapses the sample list. • Navigate through validation test categories (IQ or OQ, software or hardware) and tests. The symbols have the following meaning: • Blue document symbols indicate that qualification tests have not yet been run. • Green checkmarks indicate passed tests.
NOTE Items “inherit” the symbol (status) of their “child(s)”. For example, if a single test fails, the whole validation will be marked with a red cross (failed). A context menu appears if you right-click any item. The following functions are available: Menu Item Function Close Closes the selected validation results file. Print Displays the Print dialog box allowing you to generate a validation report. Refer to “Print (Validation)” on page 535.
NOTE The Select Data Files list also contains all electrophoretic .xad files that have been opened in the Data and Assay context. • Navigate through the samples of the selected chip data file. • Add samples to new or existing comparison files. Both the upper and the lower part provide context menus.
In the upper part of the tree view, right-clicking a xac. file name opens a context menu with the following functions Menu Item Function Save Saves the selected comparison file under its current name. Close Closes the selected comparison file. Print Displays the Print dialog box allowing you to generate various printouts of the selected comparison file. Refer to “Print (Comparison)” on page 538.
In the lower part of the tree view, right-clicking a sample name opens a context menu with the following functions: Menu Item Function Add Sample to Comparison File Adds the selected sample to the comparison file that is currently selected in the upper part of the tree view. Add Sample to Creates a new comparison file and adds the selected sample to it.
Lower Panel The lower panel is located in the lower left corner of the application window. It is available in the Data and Assay context, where you can use it to navigate through your samples. TIP To get more screen space for the Tree View Panel, you can hide the lower panel by selecting View > Lower Panel. Chip Icon If the Gel Tab, Histogram Tab (Single/Grid View), or Dot Plot Tab (Single/Grid View) is displayed, a chip icon is visible on the lower panel.
Small Gel View If the Electropherogram Tab (Single/Grid View) is displayed, the lower panel shows a small gel view (all wells). One lane of the small gel view is marked with a solid rectangle. This is the selected lane, which corresponds to a well of the chip. Clicking on a different lane will cause that lane to be selected and the electropherogram graph will update to display and highlight the associated electropherogram.
When a new chip run begins, the small gel view is blank and the first lane—the ladder well—is selected. As data is acquired, the selection rectangle around the lane will step from left to right and highlight the lane that is currently acquiring data: However, if you select a lane/well that is earlier in sequence than the current well, the highlight will no longer change as new samples are measured but will remain on the selected lane.
Tabs The main working area contains several tabs.
Instrument Tab (Grid View) Purpose Shows “live” electropherograms and/or dot plots, allowing you to watch data acquisition on two bioanalyzers in parallel.
Elements Both panels are labeled with name of the assay and the currently measured sample. For a detailed description of the “live” electropherograms and dot plots, refer to “Instrument Tab (Single View)” on page 404. Actions • Use the toolbar (see “Tile Menu” on page 362) to modify the display. • Click and drag with the mouse to zoom into the graphs. • Right-click the panels and use the context menu for further actions (see “Instrument Tab (Single View)” on page 404).
Instrument Tab (Single View) Purpose The Instrument tab lets you configure and start a chip run. During the chip run, you can watch the measurement results (raw data).
Elements The icon on the left-hand side shows the current status of the Agilent 2100 bioanalyzer: Icon Meaning A chip is detected and the instrument is ready for measurement. The chip icon depends on the type of assay. The chip icon is more than just a picture: before a chip is run, all the wells on the chip icon are black; during a chip run, a blinking white circle indicates the currently measured well. Bioanalyzer detected. Lid is open. Bioanalyzer detected. Lid is closed, but no chip is inserted.
Icon Meaning Indicates that no bioanalyzer has been detected. Check the PC’s COM port settings, the RS 232 connection cable, and make sure that the instrument is powered on. For details, refer to the Agilent 2100 Bioanalyzer Installation and Safety Guide. The following information read from the instrument is for your information: Name Meaning Name “Friendly” name of the bioanalyzer. Serial# Serial number of the bioanalyzer.
The following controls let you select, start, and control a chip run. Control Meaning COM Port Allows you to select a bioanalyzer by specifying the number of the serial port the instrument is connected to. You can also select Demo, if you want to run a demo assay without using a bioanalyzer. Assay Selection Lets you select an assay for the chip run. Only assays suitable for the inserted chip are available. See also “Assays Menu” on page 337.
Chip Setup Sub-tab On the Chip Setup sub-tab, you can specify a destination for the chip data file, and make data acquisition settings. Destination Default Select Default if you want to save the chip run in the default directory. The default directory can be defined in the Options dialog box. Refer to “Options – Data Files” on page 501. Custom Select Custom if you want to save the chip run in a different place. Clicking on the ...
Further settings can be made for flow cytometric assays: Default Enable this option if you want to use the default time for measurement. The total default time for all samples is 240s per sample. Fixed time Enable this option if you want to measure each samples within a defined time. Enter the time (in [s]) that you want to be used for each sample. The maximum measurement time per sample is calculated from the number of samples you selected.
Raw Signals Sub-tab During an electrophoretic chip run, the Raw Signals sub-tab shows an electropherogram of the currently measured sample. The name of the sample is displayed above the graph. The graph is a “live” plot of the migration time against fluorescence units (raw data, including background fluorescence, for example).
Dot Plot Sub-tab During a flow cytometric chip run, the Dot Plot sub-tab shows single events (cells) as they are detected, displayed as dots. In the coordinate system, the red and blue fluorescence value of each event can be read. The name of the currently measured sample is displayed above the graph, the number of total events and the percentage of gated events are shown below the graph. Regions (if defined) are displayed as colored rectangles.
Diagnostics Tab On the Diagnostics tab, you can select and start hardware diagnostics tests on the selected bioanalyzer. The tests check the hardware components and get the current operating status of the instrument. To perform the complete set of hardware diagnostic tests successfully, you must use unused chips or special chips from the test chip bundles (see “Running Instrument Diagnostics” on page 303).
Instrument Information At the top of the Diagnostics tab, the following information on the selected bioanalyzer is shown: Serial # Serial number of the bioanalyzer. Name “Friendly” name of the bioanalyzer. Firmware Version number of the firmware. Product ID Agilent product number of the bioanalyzer. Available Tests Columns Apply Lets you select the test for execution. Name Name of the diagnostics test. Description Brief description of the diagnostics test.
Bioanalyzer Tests Electronics Test Verifies proper functioning of all electronic boards in the bioanalyzer. Lid Sensor Test Verifies proper operation of the lid sensor, ensuring that the laser and LED are off when the lid is open. Stepper Motor Test Checks for proper movement of the stepper motor. Fan Test Checks if the fan is running at the appropriate speed. Temperature Test Checks if the temperature ramp-up speed of the heater plate is within specifications.
High-Voltage Stability Tests high voltage accuracy and stability of all 16 high voltage Test power supplies. Unused chip (DNA, RNA, or protein) required. High-Voltage Accuracy Check of the high voltage controller. Test High-Voltage Accuracy Check of channel-reference diode in transmission direction. on Load Test Autofocus Test Checks the focusing capability of the optical system. Autofocus test chip required. Laser Stability Test Measurement of stability of red laser signal.
System Leak Test Checks if the bioanalyzer is able to maintain a vacuum. Produces a test pressure of -100 mbar and monitors for changes. Cell Autofocus test chip required. Cell Autofocus Test Checks if the optical system of the bioanalyzer is correctly calibrated. Cell Autofocus test chip required. Command buttons Start/Stop Starts the selected test(s). During test execution, this button changes to Stop, which lets you abort the test(s). Select All Selects all tests displayed in the list.
Tabs (Data and Assay Context) In the Data and Assay context, the following tabs are available: • Assay Properties Tab lets you display and modify the properties of the current assay. • Chip Summary Tab allows you to edit the chip data for the current assay. • Gel Tab allows you to evaluate the results of electrophoretic assays using a gel view. • Electropherogram Tab (Single/Grid View) allows you to evaluate the results of electrophoretic assays using electropherograms.
Assay Properties Tab Purpose On this tab, you can display and modify parameters (setpoints) of the current assay or chip data file. You can also import markers and regions (flow cytometric assays only), and setpoints of other assay or chip data files (see “Importing Data” on page 256).
Flow Cytometric Assays Access The Assay Properties tab is always available in the Data and Assay context.
Elements Assay Icon This icon visualizes the assay type.
Flow Cytometric Assays Contents Apoptosis/ Red to Blue GFP (Green Fluorescence Protein) Antibody Staining/ Blue to Red Generic/ Checkout Beads siRNA Transfection Viability ▲ 421 ▼ Index
File Name Name of the assay (.xsy) file the current assay or chip data file is based on; .csy or .asy file if the basis is a Bio Sizing or Cell Fluorescence assay file. Title Name of the base assay. Location Path to the base assay file. Assay Assay type (corresponds to the Assay Icon). Gating (flow Gating direction (see also “Gating direction” on page 230): cytometric assays blue to red, or only) red to blue. Version Assay version. Modified Date the assay or chip data file was modified last.
Analysis sub-tab Shows a selection of characteristic assay parameters. Ladder sub-tab (electrophoretic assays only) Ladder peak table, one row per peak.
Fragments sub-tab (RNA assays only) Number: order in which the fragments are detected. Fragment Name: user-assigned or predefined name of the fragment. Typically 16S/23S for Prokaryote assays or 18S/28S for Eukaryote assays. Fragment start time: start time (in seconds) of the peak. Fragment end time: end time (in seconds) of the peak. The start and end times are represented on the electropherogram by diamond-shaped dots on the peak baseline in the Fragment Color.
Setpoint Explorer Lets you modify assay parameters (data analysis setpoints) globally, that is, for all samples. Click the + nodes to expand, and the – nodes to collapse branches. Setpoints that you can change are white. To edit a setpoint, double-click the value, enter the new value, and press enter. Edited values are red (until you apply them). Normal/Advanced: Switches between normal and advanced mode. Expand/Collapse: Expands/collapses all nodes.
TIP When applying modified data analysis setpoints, the software will (by default) immediately recalculate the raw data, which takes some time. Click on in the toolbar or select Don’t Analyze from the Gel Menu or Electropherogram Menu to temporarily switch off automatic data analysis while you modify setpoints.
Chip Summary Tab Purpose The Chip Summary tab shows information on the selected assay or chip data file, and lets you enter comments regarding chip, samples, and study.
Access The Chip Summary tab is available in the Data and Assay context. Elements Chip icon The chip icon indicates the assay type: DNA, RNA, Protein, or Cell. File Name Name of the chip data (.xad) file. Location Directory where the chip data file is stored. Created Creation date of the chip data file (measurement date). Modified Date when the chip data file was last modified. Software 2100 expert versions used to create and modify the chip data file. Import...
Sample Information sub-tab (electrophoretic assays) The sample table includes: Sample Name Lets you enter easy-to-remember names for your samples. Change the default names of the samples to names of your choice. Simply highlight the existing name (for example, “Sample 1”) and type a new name. Sample Comment Lets you enter a comment for each sample. Sample Name and any associated Sample Comment will appear on the printout.
Rest. Digest (DNA only) Indicating that a sample is a restriction digest allows 2100 expert to note the possible comigration of peaks. Since it is assumed that the molarities of all the fragments should be the same, any peaks or clusters having molarities that are significantly larger than the rest are flagged as potentially comigrating peaks, allowing you to examine these in more detail. See also “Data Analysis: DNA” on page 92.
Reagent Kit Lot # Lot number of the reagent kit. Chip Comments Lets you enter notes to document the chip and/or the chip run. These notes will also be printed with the report. Sample Information sub-tab (flow cytometric assays) The sample table shows you the main measurement results and allows you to enter sample names and comments. Sample Name Lets you enter easy-to-remember names for your samples. Sample Comment Lets you enter a comment for the sample.
Blue Staining Lets you enter the name of the dye that is used for blue fluorescence staining. Red Staining Lets you enter the name of the dye that is used for red fluorescence staining. Status A green check mark indicates that the sample has been measured successfully. A red cross indicates an incomplete or aborted measurement. No symbol indicates that the sample has not been measured at all. Total Events Shows the number of events detected for the sample.
Study Information sub-tab To identify your assays easily, you can add information about the study (Study Name and Study Comments), the Experimenter, the Laboratory, the Company and the Department. This can be used for data exchange with other departments or companies.
Instrument Information sub-tab This tab displays the temperature of the chip base plate recorded during the chip run. The acquisition time is displayed in relation to the temperature. NOTE The regular chip temperature should be 25 °C for flow cytometry and 30° C for electrophoresis assays. Significant differences can negatively influence your measurement results.
Standard Curve sub-tab (DNA/Protein assays only) A standard curve of migration time versus size is plotted from the sizing ladder by linear interpolation.
Standard Curve sub-tab (RNA assays only) A standard curve of mobility vs. size in logarithmic scale is plotted from the RNA sizing ladder by linear interpolation.
Calibration Curve sub-tab (protein assays only) If a standard protein was added on the chip and you have selected the option Use For Calibration on the Sample Information sub-tab, a calibration curve is calculated. See the figure above for an example. This sub-tab is only available for protein assays.
Gel Tab Purpose The Gel tab shows gel-like images of the ladder and of all samples of a chip. On sub-tabs below the gel-like image, you can find analyzed data for each individual sample. For further analysis, you can use the setpoint explorer on the right to modify data analysis setpoints for the current sample (Local tab) or for all samples (Global tab).
Access The Gel tab is always available • in the Data and Assay context if an electrophoretic chip data (.xad) file is selected, • in the Comparison context if a comparison (.xad) file or one of its samples is selected. Gel View One lane of the gel view is surrounded by a rectangle. This is the selected lane (one lane is always selected) and corresponds to a well on the chip; the chip icon on the lower panel (see “Lower Panel” on page 398) highlights the associated well.
When a new chip run starts, the gel view is blank and the first lane—the ladder well—is selected. As data is acquired, the selection rectangle around the lane will step from left to right, highlighting the lane that is currently acquiring data. However, if you select a lane that is earlier in sequence than the current lane, the highlight will no longer change as new samples are measured but will remain on the selected lane.
The dividing line between the gel graphs and the sub-tabs at the bottom can be moved in the vertical direction, giving more or less space to either area: When you right-click the gel view, the following menu appears: The following functions are available: Undo Zoom Undoes the last zoom, pan, or scale action. Undo All Undoes all zoom, pan, and scale actions. Copy Gel Puts a copy of the gel graph (of all samples) onto the clipboard.
Results sub-tab For DNA and protein assays, the Number of peaks found is shown. For protein assays, also the total relative concentration (Total Rel. Conc.) in µg/ml is shown. For RNA assays, the RNA Area, and the RNA Concentration in pg/µl or ng/µl, and the percentage of rRNA Contamination (mRNA) found in the samples is shown. Peak Table sub-tab For each peak (rows), the table shows a number of calculated values (columns).
The analyzed data depends on the assay type: • DNA Default table columns are: Peak Number The order in which the peaks were detected. (leftmost column) Second column Symbols in this column indicate the peak type. Size [bp] The number of calculated DNA base pairs. Conc. [ng/µL] The concentration in nanograms per microliter for each fragment (derived from the area/conc. relationship with the upper marker, the same for all ladder peaks).
• RNA Default table columns are: Peak Number The order in which the peaks were detected. (leftmost column) Second column Symbols in this column indicate the peak type. Time corrected area The area under the peak is corrected as a result of migration and baseline correction. Observations Additional information about the peak such as possible comigration or expected fragment indication. • Protein Default table columns are: Peak Number The order in which the peaks were detected.
Calib. Conc. [µg/ml] Calibrated concentration of the calibration protein in the standard, or of the calibrated protein in the sample. % Total The percentage of the area of the individual peak compared to the summed total area of all peaks in the sample (not including markers and system peak). Observations Additional information about the peak such as possible comigration, expected fragment indication, or calibration protein.
Calibration protein Manually set calibration protein Excluded peak Context menu When you right-click the peak table, the following menu appears: The following functions are available: Export... Opens a system dialog box allowing you to save the peak table as a .csv or .xls file. Configure Columns... Opens the Configure Columns dialog box. Copy To Clipboard Puts the peak table onto the clipboard. You can paste the table in another application such as MS Excel®.
Scale to Selected Adapts the scale of the Y axis. The selected peak is displayed at Peak maximum height. Other peaks might be cut off. (electropherogra ms only) Manually Set Lower Marker Makes the selected peak the lower marker. Manually Set Upper Marker Makes the selected peak the upper marker. Calibrate Protein Makes the selected peak the calibration protein. (protein assays only) Exclude Peak Contents Excludes the selected peak from the analysis for the sample.
Fragment Table sub-tab (RNA assays only) For each peak (rows), the table shows results for the predefined/specific fragments. Default table columns are: Fragment The order in which the fragments were detected. Number (leftmost column) Name A user-assigned or predefined name for the found fragment. Typically 16S/23S for Prokaryote assays or 18S/28S for Eukaryote assays. Start Time [s] Shows the start time for the peak in seconds. End Time [s] Shows the end time for the peak in seconds.
Area The area of the individual fragment measured in base pairs. % of total Area The percentage of the area of the individual fragment compared to the total area or RNA measured above the baseline. NOTE You can include further columns and also exclude columns from the fragment table. Right-click the heading row of the table and select Configure Columns... from the context menu. For details refer to “Configure Columns” on page 543.
Setpoint Explorer To show the setpoint explorer, click the vertical bar on the right edge of the application window: The setpoint explorer appears. You can use the setpoint explorer to modify data analysis setpoints for the current sample (Local tab) or for all samples (Global tab). Refer to “Assay Properties Tab” on page 418 and “Changing the Data Analysis” on page 111 for details on the setpoint explorer.
Electropherogram Tab (Single/Grid View) Purpose The Electropherogram tab shows electropherograms of all samples (including the ladder) of a chip, either for all (grid view) or for an individual sample (single view). Electropherograms are plots of the migration time against fluorescence units.
Single View Access The Electropherogram tab is always available • in the Data and Assay context if an electrophoretic chip data (.xad) file is selected, • in the Comparison context if a comparison (.xad) file or one of its samples is selected.
Electropherogram View In Grid View, you see electropherograms of all samples (including ladder) at the same time. The electropherogram of the selected sample is surrounded by a black rectangle. The small gel view on the lower panel (see “Lower Panel” on page 398) highlights the associated well. In Single View, you can overlay the electropherograms of different samples from one chip run.
Moving the mouse pointer over a curve in an electropherogram shows the sample name and number.
The dividing line between the electropherogram graphs and the sub-tabs can be moved in the vertical direction, giving more or less space to either area: When you right-click the electropherogram, the following menu appears: The following functions are available: Undo Zoom Undoes the last zoom, pan, or scale action. Undo All Undoes all zoom, pan, and scale actions.
Copy Puts a copy of the electropherogram into the clipboard (single-well Electropherogram view). In multi-well view, all electropherograms are copied. Save Opens a system dialog box, allowing you to save the Electropherogram electropherogram (single-well view) or all electropherograms (multi-well view) as an image in JPEG (.jpg), Windows Bitmap (.bmp), Windows Meta File (.wmf), CompuServe Graphics Interchange (.gif), or Tagged Image File (.tif) format.
When you right-click the top of a peak, the following menu appears: The following functions are available: Scale to Selected Adapts the scale of the Y axis. The selected peak is displayed at Peak maximum height. Other peaks might be cut off. Manually Set Lower Marker Makes the selected peak the lower marker. Manually Set Upper Marker Makes the selected peak the upper marker. Manually Set Calib. Protein (protein assays only) Makes the selected peak the calibration protein.
Legend sub-tab To compare samples, you can overlay electropherograms from multiple samples (single view only). Each electropherogram will then be shown in a different color, and a color legend appears on the Legend tab. This lets you easily assign curves to samples. You can change the colors of the electropherograms in the Options dialog box, see “Options – Graph Settings” on page 506.
Results, Peak Table, and Fragment Table sub-tabs The sub-tabs Results, Peak Table, and Fragment Table work exactly in the same way as they do in the gel view. For details on these sub-tabs, please refer to “Gel Tab” on page 438. Errors sub-tab Displays Code, Description and Category of any errors that occured during measurement or analysis. Most errors are the result of peaks not being located by the analysis algorithms of the software.
Histogram Tab (Single/Grid View) Purpose Histograms are plots of fluorescence intensity against the number of detected events (detected cells). For each sample on the chip, the Histogram tab shows two histograms, one for blue (left histogram) and one for red staining (right histogram), either for all 6 samples (grid view) or for an individual sample (single view).
Single View Access The Histogram tab is always available in the Data and Assay context if a flow cytometric chip data or assay file is selected. You can switch between the histogram views by choosing Single View or Grid View in the Histogram menu.
Histogram View Histograms are graphical representations of the measurement results, where the number of events is mapped to the Y axis and their fluorescence values to the X axis. The X axis can be logarithmic or linear. The resulting curves show the frequency distribution of the events in relation to their fluorescence intensity values. In the Grid View, an overview of all samples is shown.
Result Tables The result tables show a number of calculated values (columns) for each histogram. If you add or redefine markers, the data in the result tables is recalculated. If the option Hide superset curve... is disabled in the setpoint explorer (see “Assay Properties Tab” on page 418), two additional rows are displayed in the gated histogram’s result table—”CD3-APC on all events” and “CD3-APC on subset” in the example above.
% of gated Percentage of events selected by the marker in the gated histogram related to the events that have passed the gate. Mean Mean fluorescence value. StdDev Standard deviation to the mean value. %CV Coefficient of variation. GMean Geometric mean. NOTE You can exclude columns from the result tables. Right-click the heading row of the table and select Configure Columns... from the context menu. For details refer to “Configure Columns” on page 543.
Context Menus In Single View, two context menus are available. When you right-click a histogram, the following menu appears: The following functions are available: Undo Zoom Undoes the last zoom action. Undo All Undoes all zoom actions. Copy Graph To Clipboard Puts a copy of the selected histogram (blue or red) into the clipboard. Save Graph To File Opens a system dialog box allowing you to save the selected histogram (blue or red) as an image in Windows Meta File (.wmf), Windows Bitmap (.
Gate in Blue histogram Uses the selected marker of the red histogram for gating in the blue histogram (generic assays only). Show Data Points Shows/hides the data points used to generate the selected histogram. Configure Marker Opens the Configure Marker dialog box (see “Configure Marker” on page 545), allowing you to change the properties of the selected marker. Only available if a marker is selected. Delete Marker Deletes the selected marker.
Copy To Clipboard Puts the result table into the clipboard. You can paste the table in another application such as MS Excel®. If a part of the table is selected, only this selection is copied. Gate in Red/Blue Inserts a gate in the displayed gating direction (generic assays only). Histogram Only available if a marker without gate is selected in the histogram. Configure Marker... Opens the Configure Marker dialog box. Only available if a marker is selected. Delete Marker Deletes the selected marker.
Dot Plot Tab (Single/Grid View) Purpose Dot plots show single events (cells), displayed as dots. In the coordinate system, the red and blue fluorescence values of each event can be read. For each sample of the chip, one dot plot is shown, either for all 6 samples (grid view) or for an individual sample (single view).
Single View Access The Dot Plot tab is always available in the Data and Assay context if a flow cytometric chip data file is selected. You can switch between the dot plot views by choosing Single View or Grid View in the Dot Plot menu.
Dot Plot View Dot plots are graphical representations of the measurement results, where the blue fluorescence intensity is mapped to the X axis and the red fluorescence intensity is mapped to the Y axis. Both axes are logarithmic. In the Grid View, an overview of all samples is shown. The small dot plots are labeled with the sample name, and below them, the total number of events and the %-value of the covered events can be read.
Default table columns are: Region The first region (All Events) always displays the values for all detected events. For each further region (see “How to Add Regions to Dot Plots (Generic Assay only)” on page 221), a row is added to the table. XMean Mean fluorescence values in x direction. YMean Mean fluorescence values in y direction. #Events Number of events for each region added to the dot plot. % Total Percentage of events for each region added to the dot plot.
Context Menus In Single View, two context menus are available. When you right-click a dot plot, the following menu appears: The following functions are available: Undo Zoom Undoes the last zoom action. Undo All Undoes all zoom actions. Copy Graph To Clipboard Puts a copy of the selected dot plot onto the clipboard. Save Graph To File Opens a system dialog box allowing you to save the selected dot plot as an image in Windows Meta File (.wmf), Windows Bitmap (.bmp), or JPEG (.jpg) format.
When you right-click a result table, the following menu appears: The following functions are available: Export... Opens a system dialog box allowing you to save the result table as a .csv file. Configure Columns... Opens the Configure Columns dialog box. Copy To Clipboard Puts the result table into the clipboard. You can paste the table in another application such as MS Excel®. If a part of the table is selected, only this selection is copied. Configure Region... Opens the Configure Region dialog box.
Result Flagging Tab Purpose On this tab, you can define flagging rules for electrophoretic measurement results. A specific color code will be assigned to each sample with measurement results meeting a specific rule. Access The Result Flagging tab is always available in the Data and Assay context if an electrophoretic chip data or assay file is selected.
Rule List Lists all user-defined result flagging rules, and the default rule, which you cannot delete. The IF Expression of the default rule is “TRUE”. The default rule defines a color code which is assigned to samples, for which no other rule applies. The Order ID column determines the sequence in which the rules are applied to the samples. The other columns show the definitions made in the rule definition area (see below). • Normal mode All rules are applied subsequently.
• If Expression Lets you compose an expression from functions, variables, and logical operators. • Comment Lets you enter a comment for the rule. • Color Expression Lets you select a color code to be used for flagging samples that meet the rule. By selecting Gradient, you can choose two colors to specify a color gradient instead of a solid color. Help Pane Provides detailed help on the function or variable you are currently working with.
The following types are used: Return type Description int Integer number, e.g. 12 float Floating point number, e.g. 3.14159 or -1.2e-10 string Character string; string constants must be put in quotation marks, e.g. “Agilent” enum Enumeration, see int PeakFound(float Size, enum Windowtype, float WindowSize) The following functions are available: Function Description PeakFound Used to check whether certain sized fragment is found or not.
Function Description TotalArea Returns the sum of the areas of all the peaks detected in a sample. TotalConcentration Returns the sum of the concentrations of all the peaks detected in a sample. PeakAttribute Returns the attribute of given peak. PeakMax Returns the peak number of the peak having highest value in given column PeakMin Returns the peak number of the peak having the lowest value in given column. SubStr Returns a substring of a string.
Variables Variable Description Well This string variable stands for the coordinates of the well. As it evaluates to a string, it can be used in any expression where string is expected. SampleName This string variable stands for the sample name. As it evaluates to a string, it can be used in any expression where string is expected. SampleCategory This string variable stands for the category of the sample. As it evaluates to a string, it can be used in any expression where string is expected.
Operators Operators are available for comparison (=, <, <=, >, >=, <>), for arithmetical operations (+, -, *, /), and for logical operations (AND, OR, AND NOT, OR NOT).
Log Book Tab Purpose The Log Book tab shows chip run events in the run log table. For example, you can see when and by whom the chip run was performed. Also errors and problems with hardware or software are reported by the run log. Access The Log Book tab is available if a chip data (.xad) file is selected in the Data and Assay context. Run Log Table The run log table has the following columns: Description Message text describing the run log entry. Number Error number.
Source Source that triggered the run log entry: “Reader”, “Instrument” or “User Interface”. Category “Run”, “System”, “Reserved”, “Sample”, or “Assay”. User User who was logged in on Host when the run log entry was created. Host Name of the computer from which the chip run was started. Time Stamp Date and time the run log entry was created. The run log table is saved as part of the chip data (.xad) file, and you cannot delete it.
Columns... Opens a dialog box allowing you to hide, show, and re-sort columns. Column Width... Opens a dialog box allowing you to resize the selected column. Sort by Event Sorts the table by the Category column (ascending). Sort Sorts the table by the selected column. Filter... Opens the Filter Events dialog box allowing you to hide table entries matching filter criteria you can specify. A filter can hide all uncritical events, for example. Find...
Tabs (Validation Context) In the Validation context the following tabs are available: • Configuration Tab • Results Tab Contents ▲ 484 ▼ Index
Configuration Tab Purpose The Configuration tab lets you select qualification tests to be executed in a validation run. During a validation run, it shows the status and results of the tests. Available Tests Columns Apply Lets you select the qualification test for execution. Name Name of the qualification test.
Description Brief description of the qualification test. Runs Shows how many times the qualification test has been run in this validation. Status For each qualification test, the current status is shown: • Not Selected • Selected • Executing • Execution pending • Executed, passed • Executed, failed Buttons: Select All Selects all qualification tests for execution. Unselect All Deselects all qualification tests.
Results Tab Purpose The Results tab lets you view detailed results of qualification tests. The results are shown for one test at a time, but you can browse through the tests.
Test Name: ... Columns Execution # Number of execution (in case of multiple executions of the test). Date and Time Date and time the qualification test has been executed. Pass State Qualification test has passed. Qualification test has failed or was aborted. Comment Additional information on test execution. Buttons: Jumps to the results of the previous qualification test. Jumps to the results of the next qualification test.
Tabs (Comparison Context) In the Comparison context the following tabs are available: • Comparison Summary Tab • Gel Tab (see “Gel Tab” on page 438) • Electropherogram Tab (Single/Grid View) (see “Electropherogram Tab (Single/Grid View)” on page 451) Contents ▲ 489 ▼ Index
Comparison Summary Tab Purpose The Comparison Summary shows information on the selected comparison file, and lets you enter a comment regarding the comparison. Access The Comparison Summary tab is available in the Comparison context if a comparison (.xac) file or one of its samples is selected.
Elements Chip icon The chip icon indicates the assay type: DNA, RNA, or Protein. File Name Name of the comparison (.xac) file. Location Directory where the comparison file is stored. Created Creation date of the comparison file. Modified Date when the comparison file was last modified. Software 2100 expert versions used to create and modify the comparison file. Assay Class DNA1000, for example. Sample Name Name of the sample as given in the chip data (.xad) or assay (.xsy) file.
In the Comparison context also the following tabs are available: • Gel Tab (see “Gel Tab” on page 438) • Electropherogram Tab (Single/Grid View) (see “Electropherogram Tab (Single/Grid View)” on page 451) Please refer to “Comparing Samples from Different Electrophoretic Chip Runs” on page 136 for details on comparing samples from different chip runs.
Status Bar The status bar at the bottom of the 2100 expert application window shows: • System messages • Progress bar A progress bar appears, for example, during file opening or saving activities, or during a chip run. • Date and time • Status of the Auto Export, Auto Print, and Auto Run options. Disabled options are grayed out.
Dialog Boxes The 2100 expert software provides the following dialog boxes: Dialog Box Purpose General “About 2100 Expert” on page 497 To get information on bioanalyzer hardware and software. “2100 Expert – Close” on page 499 To save unsaved changes on exiting the 2100 expert application or on switching to another context. “2100 expert – End of Run” on page 511 Shows the status of the finished (or aborted) chip run. “Auto Export” on page 523 To specify options for automatic data export.
Dialog Box Purpose “Options – Advanced” on page 508 To enable automatic print, automatic export, and automatic chip run. “Page Setup” on page 555 To select the page size and format for printout. “Print (Validation)” on page 535 To set print options for validation reports and start printing. “Report Preview” on page 557 To show a page preview before printing. “Save Selected Samples ...” on page 559 To select a subset of samples for saving.
Dialog Box Purpose “Configure Marker” on page 545 To configure the marker in a histogram. “Copy Marker” on page 554 To confirm or skip the usage of the currently selected marker as a reference marker for all histograms. “Insert Existing Markers” on page 549 To insert existing markers in a histogram. “Configure Region” on page 547 To configure the region in a dot plot. “Copy Region” on page 553 To confirm or skip the usage of the currently selected region as a reference region for all dot plots.
About 2100 Expert Purpose This dialog box is used to get information about the installed bioanalyzer hardware and software. Access You can open this dialog box by selecting Help > About.
Elements Module/License Lists the installed software packages and the associated registration Key keys that were used to activate the packages. System Info Starts the System Info application of the Windows® operating system. The button is only displayed if the System Info application of Windows® is installed. Refer to the Windows® documentation. OK Closes the dialog box.
2100 Expert – Close Purpose This dialog box lets you decide whether or not to save unsaved changes. Access This dialog box appears if you try to exit the 2100 expert application (or if you try to switch between contexts) but there is unsaved data.
Elements Save changes to If you want to save changes to particular files, you can select or the following deselect individual files of the list by single-clicking them. By default, files? all files with unsaved changes are highlighted. Yes Saves changes to the selected files and quits 2100 expert (or switches to another context). No Exits the 2100 expert application (or switches to another context) without saving anything.
Options – Data Files Purpose On the Data Files tab of the Options dialog box, you can specify the names to be used for chip data file storage. Chip data files are automatically named when they are generated. Access Open this dialog box by selecting Tools > Options... and clicking the Data Files tab.
Elements You can make the following settings to specify Data File Names: Prefix The prefix you enter here will be the first part of the file name. The default file prefix is “2100 expert”. Serial Number Inserts the serial number of your Agilent 2100 bioanalyzer instrument in the file name. This is especially useful if you work with a multi-instrument system. Assay Class Inserts the assay type in the filename. For example, “DNA1000”, “Protein50”, “Apoptosis”, or “Generic”.
You can specify the directory where you want to store your chip data files (Data File Directory): Default The default directory is the “..\Data” subdirectory of the installation directory. Custom A directory of your choice. Use the Browse button to the right of the text field to open the Directory search dialog box allowing you to select the directory the data files should be stored in.
Options – Chip Alert Purpose On the Chip Alert tab of the Options dialog box, you can specify that the bioanalyzer “beeps” when a chip run is complete and the chip has to be removed from the Agilent 2100 bioanalyzer. Access Open this dialog box by selecting Tools > Options... and clicking the Chip Alert tab.
Elements Off Turns the alert sound off. Default Sound Standard sound for the chip alert. Custom Sound Allows the use of a custom sound for the chip alert. Any .wav file is allowed. Click the ... button to the right of the text field to open a dialog box to select the file. The file name will be displayed in the text field.
Options – Graph Settings Purpose You can compare samples by overlaying their electropherograms/histograms. On the Graph Settings tab of the Options dialog box you can set the signal (electropherogram/histogram) colors in overlaid graphs. Access Open this dialog box by selecting Tools > Options... and clicking the Graph Settings tab.
Elements Signal Color Contents The colored box next to Signal n (where n stands for the sample number) shows the color of the curve in the overlaid graph. Click this box to open the Color dialog box. You can select a color and assign it by clicking OK.
Options – Advanced Purpose On the Advanced tab of the Options dialog box, you can: • Set disk space limits for data storage • Set automatic chip run, automatic export, and automatic printouts. Access Open this dialog box by selecting Tools > Options... and click the Advanced tab.
Elements Limit the storage Select this option to limit the amount of disk space reserved for raw of raw data data storage. The raw data backup files (packet files, .pck) are stored backups in the “..\packets” subdirectory. The default for the Upper Limit (in MB) is 20, which corresponds to approximately 20 chip data files. Limit the storage Select this option to limit the amount of disk space reserved for the of system log system log file “SystemLogBook.log” (located in the “..\log” subdirectory).
NOTE The Auto Print and Auto Export settings are independent from the Print... and Export... commands in the File menu (see “File Menu” on page 326).
2100 expert – End of Run Purpose This dialog box indicates the end of the current chip run, and any errors associated with the run. Access The dialog box appears (accompanied by an alert sound, see “Options – Chip Alert” on page 504) when a chip run is completed, or if you have aborted a chip run.
Aborted chip run: Elements Status Shows the status of the chip run, either Run Aborted or Run Completed. File Name Name of the data file (.xad) the chip run results are saved to. Port COM port number the Instrument is connected to, or “Demo” if you have run a demo assay. Instrument Name of the bioanalyzer used for the chip run.
No. Sample number. A green check next to the sample number indicates a completed measurement, while a white cross on red ground indicates an aborted or incomplete measurement. Sample Sample names. Observation Shows how many events were detected (for flow cytometric assays), how many peaks were found (for DNA and Protein assays), RNA ratio amounts (for Total RNA assays), rRNA contamination (for mRNA assays), and the read time only for Cy5-labeled nucleic acid assays.
Open Purpose This dialog box is used to open • chip data files (.xad), assay files (.xsy), or to import markers or regions from such files, • comparison files (.xac), • validation result files (.xvd).
Access You can open this dialog box by selecting File> Open.... Elements Look in Allows you to select a drive and directory. File name Displays the name of the selected file. File of type Allows you to select the file type. Assay list Displays the chip data and assay files located in the selected directory. Information frame Displays information on the selected file. For chip data files, for example, you will see a preview of the gel image.
Export Options (Flow Cytometry) Purpose This dialog box is used to export flow cytometric chip data in different formats. Access You can open this dialog box by selecting File > Export ... with a flow cytometric chip data file (.xad) selected.
Elements Flow Cytometry Export categories: Result Tables Creates a .csv file containing result tables. All measured samples are exported to one file. Sample Data Exports text list files (.csv), one file per sample. The event data is intended for advanced users who want to closely analyze the data. Included are the time the event is measured, and the blue and red fluorescence values. FCS Data Creates FCS standard files (.fcs), one file per sample.
Histogram Images Exports the histogram images of all samples, two images (red and blue histogram) per sample. Clicking on the ... button opens a dialog box allowing you select from the following image formats: Windows Bitmap (.bmp), JPEG (.jpg), and Windows Meta File (.wmf). Multiple selections are possible. Export to XML Writes the complete .xad file contents to a structured .xml file. Export Directory settings: Default The default target directory is “..
Export Options (Electrophoresis) Purpose This dialog box is used to export electrophoretic chip data in different formats.
Access You can open this dialog box by selecting File > Export ... with an electrophoretic chip data file (.xad) selected. Elements Electrophoresis Export categories: Result Tables Creates a CSV file (.csv) containing result tables. All measured samples are exported to one file. On exporting you can: • Exclude Markers • Include Ladders • Strip off Excluded Peaks Sample Data Contents Exports text list files (.csv), one file per sample.
Gel Image Exports the gel-like images of all samples. You can export the gel view: • As one image • Each lane separately Clicking on one of the ... buttons opens a dialog box allowing you select from the following image formats: Windows Bitmap (.bmp), JPEG (.jpg), and Tagged Image File (.tif). Multiple selections are possible. Electropherogram Exports the electropherogram images of all samples, one image per Images sample. Clicking on the ...
Custom A target directory of your choice. Use the ... button right to the text field to open the Browse for folder dialog box. Create Daily Subdirectories If selected, a subdirectory with the naming format “YYYY-MM-DD” is created in the target directory and the export data is written to this directory. Buttons: Export Contents Click this button to export the selected data. For each data category, the Save As dialog box appears, where you can enter new file names and destination directories.
Auto Export Purpose To set options for automatic export, which takes place each time a chip run is complete. Access You can open this dialog box from the Options dialog box (see “Options – Advanced” on page 508) by clicking on the Settings... button next to the Auto Export check box.
Result Tables Creates a CSV file (.csv) containing result tables. All measured samples are exported to one file. Sample Data Exports text list files (.csv), one file per sample. The event data is intended for advanced users who want to closely analyze the data. Included are the time the event is measured, and the blue and red fluorescence values. FCS Data Creates FCS standard files (.fcs), one file per sample. This is the standard format for flow cytometry data.
Electrophoresis Data Export tab Result Tables Creates a CSV file (.csv) containing result tables. All measured samples are exported to one file.
Sample Data Exports text list files (.csv), one file per sample. These files are intended for advanced users who want to closely analyze the measurement data. Included are migration time and fluorescence values (raw data) of all test points. Gel Image Exports the gel-like images of all samples. You can export the gel view: • As one image • Each lane separately Clicking on one of the ... buttons opens a dialog box allowing you select from the following image formats: Windows Bitmap (.bmp), JPEG (.
Default Export Directories tab Export Directories It is advisable to specify individual target directories for Result Tables, Sample Data, FCS files, Images, and XML files. Clicking on the ... buttons next to these categories opens a system dialog box allowing you to specify the target directory for the category. Create daily subdirectories Contents If selected, subdirectories with the naming format “YYYY-MM-DD” are created in the target directories and the export data is written to these directories.
Print (Flow Cytometry) Purpose This dialog box is used to set print options for flow cytometric chip data and assay files and to start printing. Access You can open this dialog box by selecting File > Print... with a flow cytometric assay (.xsy) or chip data (.xad) file selected.
Elements Print Item Run Summary Includes chip and sample information in the printout. See “Chip Summary Tab” on page 427. Assay Details Includes assay name, path, and setpoints in the printout. See “Assay Properties Tab” on page 418. Dot Plot Summary Includes dot plot graphs in the printout. See “Dot Plot Tab (Single/Grid View)” on page 468. Dot Plot Statistics Includes dot plot result tables in the printout. See “Dot Plot Tab (Single/Grid View)” on page 468.
Save To File PDF Redirects the printout to a .pdf file. Clicking on the ... button opens a system dialog box allowing you to specify a target directory of your choice. HTML Redirects the printout to a set of .html files. Clicking on the ... button opens a system dialog box allowing you to specify a target directory of your choice. Buttons Page Setup... Opens the Page Setup dialog box, allowing you to set the page size, page orientation, and page margins. See also “Page Setup” on page 555. Printer...
Print (Electrophoresis) Purpose This dialog box is used to set print options for electrophoretic chip data or assay files and to start printing. Access You can open this dialog box by selecting File > Print... with an electrophoretic assay (.xsy) or chip data (.xad) file selected.
Elements Print Item Run Summary Includes chip and sample information in the printout. See “Chip Summary Tab” on page 427. Assay Details Includes assay name, path, and setpoints in the printout. See “Assay Properties Tab” on page 418. Electropherogram Includes electropherogram graphs in the printout. See “Electropherogram Tab (Single/Grid View)” on page 451. Gel Like Includes a gel-like image (all samples) in the printout. See “Gel Tab” on page 438.
Options Select how many electropherograms to print per page. Affects the printout only if Electropherogram is selected under Print Item. You can also: • Exclude Markers • Include Ladder Save To File PDF Redirects the printout to a .pdf file. Clicking on the ... button opens a system dialog box allowing you to specify a target directory of your choice. HTML Redirects the printout to a set of .html files. Clicking on the ...
Cancel Exits the dialog box without printing. Print/Save If you selected any option under Save To File, the button is labeled Save, otherwise Print. Print starts printing. Save starts writing the printout to .pdf and/or .html files.
Print (Validation) Purpose This dialog box is used to set print options for validation reports and to start printing. Access You can open this dialog box by selecting File > Print... with a validation (.xvd) file selected in the Validation Context.
Elements Print Item IQ SW Includes the results of all installation qualification tests for the 2100 expert system software. IQ HW Includes the results of all installation qualification tests on the Agilent 2100 Bioanalyzer hardware. OQ SW Includes the results of all operational qualification tests for the 2100 expert system software. OQ SW Includes the results of all operational qualification tests on the Agilent 2100 Bioanalyzer hardware. Save To File PDF Redirects the printout to a .pdf file.
Printer... Opens the Print Setup dialog box, allowing you to select a printer, and set the page size and page orientation. Preview Opens the Report Preview dialog box displaying a preview of all pages that will be printed. See also “Report Preview” on page 557. Cancel Exits the dialog box without printing. Print/Save If you selected any option under Save To File, the button is labeled Save, otherwise Print. Print starts printing. Save starts writing the printout to .pdf and/or .html files.
Print (Comparison) Purpose This dialog box is used to set print options for comparison reports and to start printing. Access You can open this dialog box by selecting File > Print... with a comparison (.xac) file selected in the Comparison Context.
Elements Print Item Comparison Summary Includes information on the comparison file in the printout. Assay Details Includes assay name, path, and setpoints in the printout. See “Assay Properties Tab” on page 418. Electropherogram Includes electropherogram graphs in the printout. See “Electropherogram Tab (Single/Grid View)” on page 451. Gel Like Includes a gel-like image (all samples) in the printout. See “Gel Tab” on page 438. Result Tables Includes result tables in the printout.
Save To File PDF Redirects the printout to a .pdf file. Clicking on the ... button opens a system dialog box allowing you to specify a target directory of your choice. HTML Redirects the printout to a set of .html files. Clicking on the ... button opens a system dialog box allowing you to specify a target directory of your choice. Buttons Page Setup... Opens the Page Setup dialog box, allowing you to set the page size, page orientation, and page margins. See also “Page Setup” on page 555. Printer...
Auto Print Purpose To set options for automatic printouts, that are to be generated each time a chip run is complete. Access You can open this dialog box from the Options dialog box (see “Options – Advanced” on page 508) by clicking on the Settings... button next to the Auto Print checkbox.
Elements Print Item Assay Details Includes assay name, path, and setpoints in the printout. See “Assay Properties Tab” on page 418. Run Summary (for Includes chip and sample information in the printout. See “Chip Flow Cytometry Summary Tab” on page 427. data) Sample Data Includes sample data in the automatic printout. This data depends on the assay type: electropherogram graphs and gel-like images, or histogram and dot plot graphs. Select how many electropherograms/histograms/dot plots to print per page.
Configure Columns Purpose This dialog box is used to show or hide columns of tables. Access You can display this dialog box by opening the context menu in a result table on the Dot Plot, Histogram, Gel, or Electropherogram tab, and selecting Configure Columns....
Elements Available This list on the left shows you all available but currently not displayed columns. Displayed This list on the right shows you all columns that are currently displayed. The sequence in this list is related to the sequence of the columns. Shifts the selected entry form the left list to the right. The selected entry will be displayed in the result table at the corresponding position. Shifts all entries of the left list to the right.
Configure Marker Purpose This dialog box is used to set the properties of the selected marker in a histogram. Access You can open this dialog by double-clicking on a marker or by double-clicking the corresponding row of the result table in a histogram.
Elements Name Enter a name for the marker. Easy-to-remember names, for example the used dye, make it easier to identify the marker. Lower Value/ Upper Value Enter fluorescence values for the lower level (left line of the marker) and for the upper level (right line of the marker). These lines define a range of relative fluorescence that you can use for gating. Color Click the ... button to display the Color dialog box where you can select a color for the marker and assign it by clicking OK.
Configure Region Purpose This dialog box is used to set the properties of the selected region in a dot plot. Access You can open this dialog box by double-clicking a region or by double-clicking the corresponding row of the result table of a dot plot. Elements Name Contents Enter the name of the region. Easy-to-remember names, for example the color, makes it easier to identify the region.
Top, Bottom, Left, The four values define a rectangular region. You can change the size Right of the region by entering different values or by dragging the borders with the mouse. Region Color Contents Click this button to open the Color dialog box where you can select a color for the region and assign it by clicking OK.
Insert Existing Markers Purpose This dialog box is used to add existing markers to histograms. Access When a histogram is displayed in single view (see “Histogram Tab (Single/Grid View)” on page 460), click the button in the toolbar (see “Data and Assay Context – Flow Cytometry Toolbar” on page 376) to open this dialog box. Description All existing regions are listed except the ones that are already used by the current histogram.
Elements Existing Markers List of available markers for the current histogram. Information frame In the right part of the dialog box, the lower and upper limits of the selected marker are displayed. Insert Marker Click this button to insert the selected marker in the current histogram. If you change the properties of a marker, the properties will change accordingly in all other histograms that use this marker.
Insert Existing Region Purpose This dialog box is used to add an existing region to a dot plot. Access When a dot plot is displayed in single view (see “Dot Plot Tab (Single/Grid View)” on page 468), click the button in the toolbar (see “Data and Assay Context – Flow Cytometry Toolbar” on page 376) to open this dialog box.
Description All existing regions are listed except the ones that are already used by the current dot plot. Existing Regions List of available regions for the current dot plot. Information Frame In the right-hand part of the dialog box, the properties of the selected region are displayed. The x and y coordinates correspond to the region’s upper left corner. Insert Region Click this button to insert the selected region in the current dot plot.
Copy Region Purpose To confirm or skip the usage of the currently selected region as a reference region for all samples. Access The dialog box is displayed when you click the Insert region into all dot plots... button . Elements The inserted region can be used as a reference region. When you change the reference region, all inserted regions in the samples will also be changed. Yes Uses the selected region as the reference region.
Copy Marker Purpose To confirm or skip the usage of the currently selected marker as a reference marker for all samples. Access The dialog box is displayed when you click the Insert selected marker into all histograms button . Elements The currently selected marker can be used as a reference marker. If you change the reference marker, all inserted markers in the samples will also be changed. Yes Click Yes to use the currently selected marker as the reference.
Page Setup Purpose This dialog box lets you change the layout of the printed page. Elements Size Allows you to select the paper format. Source Allows you to specify the tray from which the paper is to be taken. The list contents depend on the selected printer.
Portrait Prints data in portrait orientation. Landscape Prints data in landscape orientation. Margins Defines the borders of the page that you want to print. The available settings depend on the paper size. Printer... Opens a dialog box allowing you to select a printer.
Report Preview Purpose This window is used to display a preview of all pages that will be printed. Access You can open this window by clicking the Preview button in the Print dialog boxes.
Elements You can browse through the print preview and modify the display using the functions in the toolbar: Table of Contents Displays a navigation panel at the left border of the window. Print... Click this button to open a system dialog box allowing you to select a printer and start printing. Copy Puts the text of current page on the clipboard. Find Lets you search the printout for any text string. Single Page Switches to single-page preview. Multiple Pages Switches to multi-page preview.
Save Selected Samples ... Purpose This dialog box is used to select a subset of the current .xad file’s samples for saving. Access If a .xad file selected, you can open this dialog box in the Data and Assay context by selecting Save Selected Sample... from the File menu.
Elements Data File Name of the chip data (.xad) file you selected for selective saving. Location Path to the Data File. ID Sample number. Sample Name Sample name. Comment Comment for the sample. Category Sample type: • Sample • Ladder (electrophoretic assays only) Selected All samples that you select here will be written to a new .xad file. Note that you cannot deselect the Ladder (electrophoretic assays only). Apply Opens the Save Select Sample dialog box allowing you to save a new .xad or .
System Log Viewer Purpose The system log table shows system-wide events. For example, you can see who started the 2100 expert software and when. Also errors and problems with hardware or software are reported by the system log.
Access You can open this dialog box by selecting System Log from the Tools menu. System Log Table The system log table has the following columns: Description Message text describing the system log entry. Number Error number. Source Source that triggered the system log entry: “Reader”, “Instrument” or “User Interface”. Category “Run”, “System”, “Reserved”, “Sample”, or “Assay”. User User who was logged in on Host when the log entry was created.
Context Menu Right-clicking on the system log table opens a context menu: Hide Column Hides the selected column. Show All Columns Shows all columns. Columns... Opens a dialog box allowing you to hide, show, and re-sort columns. Column Width... Opens a dialog box allowing you to resize the selected column. Sort by Event Sorts the table by the Category column (ascending). Sort Sorts the table by the selected column. Filter...
Find... Opens a dialog box allowing you to search the table for any string. Export... Opens a dialog box allowing you to export the system log table (or parts of it) as an .html or .txt file. Toolbar The System Log Viewer window provides a toolbar that works in the same way as the Log Book toolbar, see “Data and Assay Context – Log Book Toolbar” on page 382.
Shortcuts and Mouse Actions You can enter data and commands either by using the keyboard or the mouse. • Shortcuts can be used to enter commands very quickly using the keyboard. • Mouse actions are required for making selections, and convenient for selecting menu commands, pressing buttons etc. Shortcuts You can use standard Windows® shortcuts such as Ctrl+C, and shortcuts specific to the 2100 expert application.
Alt Shortcuts Press and hold down the Alt key while pressing another key: Shortcut Function Alt+f Opens the File Menu. Alt+c Opens the Context Menu. Alt+v Opens the View Menu. Alt+a Opens the Assays Menu. Alt+t Opens the Tools Menu. Alt+w Opens the Windows Menu. Alt+h Opens the Help Menu. Alt+F4 Exits the 2100 expert application. Function Keys These are the function keys located at the top of your keyboard (F1 – F12).
Arrow Keys The arrow keys are located to the left of the numerical key pad on your keyboard. Shortcut Function → Moves the input focus to the right, or switches to the next tab. ← Moves the input focus to the left, or switches to the previous tab. ↑ Moves the input focus upwards, for example in the Tree View Panel. ↓ Moves the input focus downwards, for example in the Tree View Panel. Tab Moves the input focus to the next input item, for example to the next cell of a result table.
Mouse Actions The mouse lets you make efficient use of 2100 expert’s graphical user interface. Left-click Result In Toolbars Activates the function associated with the button. In Tree View Panel Data and Assay context: Selects a file, or displays the grid view (All Samples), or the single view of an electropherogram, a gel-like image, a histogram, or a dot plot. Instrument context: Selects a bioanalyzer or switches to the grid view (All Instruments).
Left-click Result In Single View Histograms: selects a marker. Dot plots: selects a region. At the right edge of the window on electropherograms and gel view: shows/hides the setpoint explorer. Gel view: selects a sample. In tables (peak table, fragment table Selects a table cell. etc.) In setpoint explorer Contents Selects a setpoint.
Double left-click Result In Single View On markers in histograms: opens the Configure Marker dialog box. On region borders in dot plots: opens the Configure Region dialog box. On gel-like images, electropherograms, histograms, and dot plots: undoes the last zoom action. In Grid View Displays the instrument, electropherogram, histogram, or dot plots in single view. In setpoint explorer On value: switches to editing mode (if setpoint can be modified).
Right-click Result In Tree View Panel Data and Assay context: selects a file or sample, and opens a context menu allowing you to save, close, or print the file. Instrument context: selects a bioanalyzer or switches to the grid view (All Instruments). Comparison context: adds or removes the selected sample from a comparison file. In Grid View Data and Assay context: selects a sample. Electropherograms: displays a context menu with several items for evaluating and editing the selected sample.
Drag Result In Single View Zooms into the graph. In electropherograms, also pan and scale operations are possible. Histograms: The mouse pointer changes to a pointing hand that lets you move the marker horizontally. Dot plots: At the region corners, the mouse pointer changes to a double arrow that lets you enlarge or reduce the region. In Grid View Data and Assay context: zooms into electropherograms. Pan and scale operations are also possible. Instrument context: zooms into the graph.
Products, Spare Parts, and Accessories To buy the following products, spare parts and accessories for the Agilent 2100 bioanalyzer, please refer to the Agilent Online Store: http://www.agilent.com/home/buyonline.html Bundles • G2940CA – Agilent 2100 bioanalyzer desktop system Includes Agilent 2100 bioanalyzer, Compaq desktop PC, color printer, system software, vortexer, and accessories. Cartridge and license must be purchased separately.
• G2947CA – Agilent 2100 bioanalyzer electrophoresis set Includes test chip kit, electrode cartridge, license key for electrophoresis assays, and start-up service. • G2948CA – Agilent 2100 bioanalyzer flow cytometry set Includes checkout kit, test chip kit, pressure cartridge, license key for flow cytometry assays, and start-up service.
• G2938-81605 – RS 232 cable Communication cable PC – instrument • 2110-0007 – Fuse Two power supply fuses are needed for the G2938C bioanalyzer • G2938-81610 – Multiport cable for rocketport card • 5042-1398 – Adjustable Clip for use with luer lock syringe • 5065-4401 – Chip Priming Station including gasket kit and adjustable clip • G2938-68716 – Gasket Kit Includes spare parts for the chip priming station: 1 plastic adapter, 1 ring and 10 gaskets • 5065-4428 – IKA Vortexer (115V) Must be ordered at IKA •
Glossary This glossary explains terms in context with flow cytometry, electrophoresis, and software or hardware of the Agilent 2100 bioanalyzer. A ASY file In Bio Sizing electrophoretic assays were stored as .asy files. 2100 expert can import .asy files. See also XSY file. B Baseline A baseline is established just after the First Peak Time setpoint. After the overall baseline is established, a local baseline is calculated for each peak to compensate for baseline drift.
The figure below shows baselines established for DNA assay peaks. Peaks for DNA and protein assays are determined on a peak-by-peak basis (the overall baseline is shown).
The figure below shows baselines established for Total RNA assay fragments. Total RNA fragments are determined on a peak-by-peak basis and an overall baseline is shown from the start to end time.
The figure below shows baselines established for an mRNA assay. mRNA fragments are determined on a peak-by-peak basis and an overall baseline is shown from the start to end time.
NOTE With RNA assays, you can move the lines marking the start and end points for data analysis (shown by the long-dashed vertical green lines) which will adjust the entire baseline for calculation of the area of the total sample. Baseline Plateau This setpoint (found in the setpoint explorer) rejects brief, low slope areas such as at peaks and between non-baseline-resolved peaks.
C CAD file In Cell Fluorescence flow cytometric chip runs were stored as .cad files. 2100 expert can import .cad files. See also XAD file. Center Point After locating a start point, the peak find algorithm looks for the first negative slope value and saves the previous point as the center. If the value of the center point is less than the Minimum Peak Height, the algorithm starts looking for a new peak. CLD file In Bio Sizing electrophoretic chip runs were stored as .cld files. 2100 expert can import .
D Data Filtering The first step 2100 expert takes in analyzing raw data is to apply data filtering. Data filtering is done by means of a polynomial “filter” that is applied to the raw data. The setting for the Polynomial Order in the setpoint explorer determines the amount of data to be applied: the smaller the number, the more data that is applied and the more filtering that takes place. Data Points Data points are 0.05 seconds apart.
Electrokinetic forces Electrokinetic forces are used to move, switch and separate the samples. Active control over voltage gradients directs the movement of materials using the phenomenon of electrophoretic flow. Electroosmotic Flow A phenomenon that results from an electrical double layer formed by ions in the fluid and surface electrical charges immobilized on the capillary walls. When an electric field is applied, the bulk solution moves towards one of the electrodes.
End Point The peak find algorithm looks for a leveling off when the value of the slope is less than the value set for the slope threshold. This is considered to be the end point of the peak. With RNA assays, individual peak end times can be moved manually by dragging the diamond-shaped end points shown in the single-well display. End Time This setpoint determines the time after the start of a run before which the last peak or fragment will be located (any peaks appearing after this time are ignored).
Firmware The firmware is a program to control the hardware of the Agilent 2100 bioanalyzer. It is downloaded from your computer to the Agilent 2100 bioanalyzer and controls, among others, data transfer or the measurement procedures. Flow Cytometry A method to detect cells with certain properties. In a continuous stream, stained cells pass through a light beam. The emitted fluorescence is used for counting and differentiation.
HTML file HTML (Hyper Text Markup Language) is the authoring language used to create documents on the World Wide Web. HTML defines the page structure, fonts, graphic elements and hypertext links to other documents on the Web. J JPG file Joint Photographic Experts Group Image File. A JPEG file is a compressed raster or bitmapped graphic image. When a JPEG is created, a range of compression qualities may be considered.
Lab-on-a-chip technology means downsizing of analytical techniques from lab-scale to chip-scale: • using techniques like electrophoresis, chromatography, and sieving. • with fluorescence, absorbance, and MS detection. • with a higher degree of automation, integrating multiple steps of a complex protocol into a miniaturized system. Virtually any biochemical testing that can be done in a laboratory can theoretically be done on a chip. Ladder Each electrophoretic LabChip Reagent kit contains a ladder.
M Microfluidics The movement of liquids through micro-fabricated structures by means of electrical fields or pressure/vacuum, holding the promise of greater functionality with significantly improved reliability: • small glass or plastic devices with micro-channels as experimental platform • active control of fluids without moving parts on-chip through miniature electrodes or pumps controlled by software scripts • emulation of conventional liquid pumps, valves, dispensers, reactors, separation systems, etc.
Miniaturized laboratories on a microchip Expression used to describe lab-on-a-chip technology. Minimum Peak Height The Minimum Peak Height value determines whether or not a peak is kept. For each peak, the difference between start point value and center point value must be greater than the Minimum Peak Height value. This setting is chosen in the setpoint explorer. Minimum Peak Width The Min Peak Width value determines whether or not a peak is kept.
PDF file PDF (Portable Document Format) is a file format created by Adobe Systems Incorporated that preserves all of the fonts, formatting, colors, and graphics of any source document, regardless of the software and computer platform used to create it. Peak Baseline A local peak baseline is calculated for each peak. For isolated peaks, the local peak baseline is simply a straight line connecting the start point with the end point.
Peak Height The value at the center point of the peak minus the local baseline start value. Point-to-Point Fit This curve fit is composed of line segments between each pair of data points that are used to interpolate data between those points. Polynomial Filter The first step 2100 expert takes in analyzing the raw data is to apply data filtering. Data filtering is done by means of a polynomial “filter” that is applied to the raw data.
Priming Station Consists of a chip holder that has a syringe mounted on the lid that seals over the chip. The syringe is used to force the buffer solution loaded into the well marked “G” with a circle around it into all the passageways inside the chip prior to running it in the bioanalyzer.
R Raw data backup file See PCK file. S Serial port The serial ports (COM ports) are used to connect your computer with the Agilent 2100 bioanalyzer. The number of available ports depends on the computer you use. Slope Threshold The Slope Threshold setpoint represents the amount of change in fluorescence units over time required to indicate that a peak has occurred. Changing this setpoint may cause certain peaks that were previously detected to be ignored.
Start Time This setting determines the time after which the first peak or fragment will be located (any peaks appearing before this time are ignored). In RNA and Protein assays, the start time is shown on the single view display as a long-dashed vertical green line (note that this is true for protein assays when analysis is on; the start time is shown as a solid green line when analysis is off for protein assays).
U Upper Marker An internal standard that is added to a DNA or Protein sample in a well to assist in determining size and concentration of the sample. The upper marker is the same as the last peak found in the sizing ladder. W WAV file A type of computer file used to store a sound digitally. WMF file Windows Metafile. Windows metafile documents can contain any mix of vector and raster (or bitmapped) information to describe the contents of an image.
XLS file Microsoft Excel spreadsheet file. XML file Extensible Markup Language files. XML is the Extensible Markup Language, a system for defining specialized markup languages that are used to transmit formatted data. XML is conceptually related to HTML, but XML is not itself a markup language. Rather it is a metalanguage, a language used to create other specialized languages. 2100 expert uses the XML format to: • export chip data • save and load result flagging rules. XSY file 2100 expert assay file.
Z Zero Baseline All electropherograms produced with the bioanalyzer show some amount of background fluorescence. By default, the 2100 expert software enables the zero baseline function. Enabling this setting offsets the graphs shown for the individual wells but does not affect analysis. The mean of 100 points before the baseline time (derived when calculating well noise) is used as the zero baseline value.
To remove the zeroing, disable the Zero Baseline box in the setpoint explorer (baseline calculation under Global and Advanced setting).
Index A Accessories, 573 Adding regions, 221 Agilent Online Store, 573 Alert sound, 296 Antibody staining, 230 APC, 159 Apoptosis assays, 235 Assay setpoints, 249 Assays Creating new, 251 Generic, 168 Opening, 252 Predefined, 166 Stopping, 77, 187 Auto Export, 301, 509 Auto Print, 301, 509 Auto Run, 301 B Base pair, 588 Bioanalyzer manuals, 9 Bioanalyzer tests, 303 Bubbles, how to avoid, 178 Contents C Calcein, 158 Capillary electrophoresis, 589 Cartridges, 47 CBNF, 159 Cell detection, 160 Chip reagents,
D Data specify file names and directories, 294 Data analysis setpoints, 111 data analysis setpoints, 249 Data files, 247 Data points, 86, 195 Dividing line, 455 Documentation, related, 9 Dot Plot generating, 164 Dyes, 158 E Electrode cartridge, 48 Electrodes, 50, 52 Electrokinetic forces, 583 Electroosmotic Flow, 583 Electrophoresis, 583 Electrophoretic flow, 583 Errors, 449, 459 Event, 161 Excel, 446, 467 Exporting automatically, 509 F FCS Express, 517 Fluorescence dye, 56 Contents G Gates working with,
Log Run log, 287 System Log, 287 M Manual integration, 124 Marker, 200 configuring, 209 copying, 212 inserting, 207 limits, 211 removing, 208 Micro-channels, 56 Microfluidics, 588 Molarity (nM), results table, 443 Molecular separation techniques, 589 Molecular weight, 588 Multi channel mode, 83, 193 N Navigation, 84, 194 O Opening assays, 252 Operational qualification, 312 OQ, 312 Overlaid histograms, 298 Overlay histograms, 214 color and scale, 216 Contents P Peak number, results table, 443 Peak type, 44
Z Zoom Dot Plot, 86, 195 Histogram, 86, 195 Undo, 87, 196 S Scale overlaid histograms, 216 Setpoint Explorer, 425 Setpoints, 249 Show data points, 88, 197 Sieving polymer, 56 Single channel mode, 83, 193 siRNA Transfection Viability, 167 Size (bp), results table, 443 Sound, 296 Spare parts, 573 Staining cells, 158 Starting an assay, 72, 182 Stop assay, 77, 187 Storage limit, 301 System Log, 287 SYTO16, 158 T Tagged Image File Format, 343, 371, 441 U Undo zoom, 87, 196 W Windows Bitmap, 343, 371, 441 Window
